Process for producing sugar chain asparagine derivative

ABSTRACT

The present invention provides a process for preparing a sugar chain asparagine derivative. According to the process, various isolated sugar chain asparagine derivatives useful in the field of the development of pharmaceuticals and the like can be conveniently obtained in a large amount as compared to that of the prior art. The present invention also provides a process for preparing a sugar chain asparagine and a process for preparing a sugar chain via a step for preparing a sugar chain asparagine derivative. The present invention further provides a novel sugar chain asparagine derivative, a sugar chain asparagine and a sugar chain.

TECHNICAL FIELD

The present invention relates to a process for preparing a sugar chainasparagine derivative and a sugar chain asparagine derivative.

BACKGROUND ART

Conventionally, a technique for degrading a sugar chain by a glycosidaseto derivatize the sugar chain has been utilized in the analyticalstudies on a several milligram scale such as structural analysis of asugar chain. However, since derivatives of individual sugar chains couldnot be obtained in a large amount, development of the studies on a gramscale has been retarded. Therefore, it has been difficult to apply asugar chain derivative in the synthesis studies such as the manufactureof pharmaceuticals.

On the other hand, it has been known that a glycopeptide is obtained ina large amount from an egg yolk (Biochimica et Biophysica Acta 1335(1997) p 23–32). However, there has not been reported a case where afluorenylmethoxycarbonylated (Fmoc-)sugar chain derivative of a compound1 shown in FIG. 1, or a series of compounds having deletions of severalsugar residues such as sialic acid or galactose on one of non-reducingterminals of a branched sugar chain in the compound 1, are obtained in alarge amount. In addition, there are some cases where several sugarchains are isolated in a small amount from a protein or the like inhuman blood. However, when the sugar chain is employed in themanufacture of a pharmaceutical, there is a risk of allowing thepharmaceutical to contaminate AIDS virus, hepatitis viruses or the like.Therefore, there has been a controversy over the technique of applyingthe sugar chain to a pharmaceutical.

Meanwhile, there are numerous examples of processes for preparing sugarchains of which branched moieties have the same structure for a branchedsugar chain. As conventional techniques, there are three kinds ofprocesses. A first is a process for isolating and purifying anasparagine type sugar chain complex from a naturally occurringglycoprotein. Representative examples of this type are those reported inT. Tamura, et al., Anal. Biochem., 1994, 216, p 335–344, V. H. Thomas,et al., Carbohydr. Res., 1998, 306, p 387–400, K. G. Rice, et al.,Biochemistry, 1993, 32, p 7264–7270 and the like. The advantage of theseprocesses is that the synthesis of the sugar chain is not necessary.However, there are several defects. For instance, the sugar chainderived from the above glycoprotein is obtained as a mixture of thesugar chains having random deletions of several sugar residues innon-reducing terminal moieties, and the sugar chains contained in theabove mixture are similar to each other in their physical and chemicalcharacteristics, so that it is very difficult to separate intoindividual sugar chains, thereby making it substantially impossible toobtain a single sugar chain in a large amount. Also, there is a casewhere a sugar chain has been isolated from a protein in human blood(isolation from Fibrinogen: C. H. Hokke, et al., Carbohydr. Res., 305(1997), p 463–468, isolation from Human Serum Transferrin: M. Mizuno et.al., J. Am. Chem. Soc., 1999, 121, p 284–290) in order to obtain thesugar chain in a relatively large amount. As mentioned above, a proteinin human blood must be handled carefully because the protein may becontaminated with an AIDS virus or hepatitis viruses. Therefore, it isdifficult to utilize the resulting sugar chains and derivatives thereofin the development of pharmaceuticals. Even if the sugar chains wereobtained in a large amount, their structures are limited, and any caseswhere sugar chains and derivatives thereof having many kinds ofstructures are obtained do not substantially exist.

In K. G. Rice, et al., Biochemistry, 1993, 32, p 7264–7270, or Rice, etal., Neoglycoconjugate, Academic Press, 1994, ISBN 0-12-440585-1 p286–321, a sugar residue is removed from a non-reducing terminal of asugar chain with a glycosidase. However, since a sugar chain having asingle structure used as a raw material cannot be obtained in a largeamount, the process can only be carried out on an analytical scale. E.Meinjohanns (J. Chem. Soc. Perkin Trans 1, 1998, p 549–560) et al. haveobtained a compound 56 shown in FIG. 5 from a bovine fetuin(bovine-derived glycoprotein) and then synthesized a compound 10 shownin FIG. 1 via a compound 33 shown in FIG. 3. In order to obtain thecompound 56, which is a first raw material, a hydrazine degradationreaction has been utilized. This hydrazine is highly toxic, so thatthere is a problem in safety when the resulting sugar chain derivativeis applied to a pharmaceutical, due to the contamination of a traceamount of the hydrazine. In addition, the sugar chain derivatives of thecompounds 56, 33 and 10 to which sialic acid is not bound can beobtained only in a small amount.

A second process is a process of synthesizing a sugar chain chemically.Currently, a construction of about 10 sugars prepared by combiningmonosaccharides according to a chemical synthesis process can be made asshown in a reported case of J. Seifert et al., Angew Chem Int. Ed. 2000,39, p 531–534. The advantage of this process is that all of the sugarchain derivatives can be theoretically obtained. However, since itspreparation steps are enormous, there is a defect that a synthesis in alarge amount is difficult. In addition, even in case where a sugar chainin which about 10 sugar residues are bound is synthesized in an amountof several milligrams, a time period of as long as about one year isrequired. While there have been so far some cases where several sugarchains are synthesized chemically, most of the cases actually couldsynthesize the intended sugar chain only in an amount as small asseveral milligrams.

A third process is a process of synthesizing a sugar chain by combiningan enzymatic reaction and a chemical reaction. As a representativeexample, there is a process as reported by Carlo Unverzagt, Angew ChemInt. Ed. 1996, 35, p 2350–2353. This process employs a technique inwhich a sugar chain is constructed to a certain length by chemicalsynthesis, and thereafter a sugar residue is added to the sugar chain byan enzymatic reaction, thereby extending the sugar chain. However, sincethe enzyme used in the chain extension has substrate specificity, thekinds of sugar which can be introduced into the sugar chain are limited.In addition, since the preparation steps are enormous in the chemicalsynthesis, a large-scale synthesis is difficult, so that the finalproduct can be obtained only in a small amount. Alternatively, in C. H.Lin et al. (Bioorganic & Medicinal Chemistry, 1995, p 1625–1630), asialyloligoglycopeptide is obtained from an egg yolk by employing aprocess reported by M. Koketsu et al. (J. Carbohydrate Chemistry, 1995,14(6), p 833–841), and the structure of the non-reducing terminalmoieties of the sugar chain is modified with a glycosidase and a sugartransferase. A sugar chain having only one asparagine (Asn) residuebound to its non-reducing terminal moiety is shown in a drawing of thisarticle. However, according to the process reported in J. CarbohydrateChemistry, 1995, 14(6), p 833–841, a mixture of sugar chains each havingon average about 2.5 amino acids other than asparagine, such as lysine,which are bound to a non-reducing terminal of the sugar chain.Therefore, a sugar chain derivative cannot be obtained as a singlecompound. Also, there is not suggested obtainment of individualderivatives of the compounds having arbitrary deletions of the sugarresidues in the branched sugar chain of the branching sugar chains in alarge amount. An article by C. H. Lin et al. does not give any evidencethat a sugar chain is obtained as a single product. Y. Ichikawa mentionsin Glycopeptide and Related Compounds (Marcel Dekker, Inc., 1997, ISBN0-8247-9531-8, p. 79–205) that if the trifurcated sugar chain complex issequentially treated from a terminal with a glycosidase, sugar chainscan be sequentially removed from a non-reducing terminal of the sugarchain, thereby giving various sugar chain derivatives. However, how theindividual sugar chains are separated after the enzymatic treatment isnot described, and the synthesis is limited only to those having uniformbranching. Therefore, even with this process, it is thought that theindividual derivatives of the compounds having arbitrary deletions ofsugar residues in the branched sugar chain of the branching sugar chainscannot be obtained in a large amount.

DISCLOSURE OF INVENTION

An object of the present invention is to provide a process for preparinga sugar chain asparagine derivative, whereby various isolated sugarchain asparagine derivatives useful in the field of the development ofpharmaceuticals and the like can be conveniently obtained in a largeamount as compared to those of the prior art. Another object of thepresent invention is to provide a process for preparing a sugar chainasparagine and a process for preparing a sugar chain via a step forpreparing a sugar chain asparagine derivative, whereby various isolatedsugar chain asparagines and sugar chains, which are useful as much assugar chain asparagine derivatives, can be conveniently obtained in alarge amount as compared to those of the prior art. Still another objectof the present invention is to provide a novel sugar chain asparaginederivative, a sugar chain asparagine and a sugar chain.

Concretely, the present invention relates to:

(1) a process for preparing a sugar chain aspargine derivative derivedfrom a sugar chain asparagine, comprising the steps of:

-   (a) introducing a fat-soluble protecting group into the sugar chain    aspargine contained in a mixture of one or more sugar chain    asparagines, to give a mixture of sugar chain asparagine    derivatives; and-   (b) subjecting the mixture of sugar chain asparagine derivatives or    a mixture obtainable by hydrolyzing a sugar chain asparagine    derivative contained in the mixture of sugar chain asparagine    derivatives to chromatography, to separate each of the sugar chain    asparagine derivatives therefrom;    (2) the process according to the above (1), further comprising the    step of (b′): hydrolyzing the sugar chain asparagine derivative    separated in step (b) with a glycosidase;    (3) the process according to the above (1) or (2), wherein the    mixture of one or more sugar chain asparagines comprises:

and/or a compound having one or more deletions of sugar residues in theabove compound;(4) the process according to any one of the above (1) to (3), whereinthe fat-soluble protecting group is fluorenylmethoxycarbonyl (Fmoc)group;(5) the process according to any one of the above (1) to (3), whereinthe step (a) is a step of introducing Fmoc group into the sugar chainaspargine contained in a mixture of one or more sugar chain asparagineshaving a sialic acid residue at a non-reducing terminal, and introducingbenzyl group into the sialic acid residue, to give a mixture of sugarchain asparagine derivatives;(6) a process for preparing a sugar chain aspargine, comprising thesteps of:

-   (a) introducing a fat-soluble protecting group into a sugar chain    aspargine contained in a mixture of one or more sugar chain    asparagines, to give a mixture of sugar chain asparagine    derivatives;-   (b) subjecting the mixture of sugar chain asparagine derivatives or    a mixture obtainable by hydrolyzing a sugar chain asparagine    derivative contained in the mixture of sugar chain asparagine    derivatives to chromatography, to separate each of the sugar chain    asparagine derivatives therefrom; and-   (c) removing the protecting group from the sugar chain asparagine    derivative separated in the step (b), to give the sugar chain    asparagine;    (7) the process according to the above (6), further comprising the    step of:-   (b′) hydrolyzing the sugar chain asparagine derivative separated in    step (b) with a glycosidase; and/or-   (c′) hydrolyzing the sugar chain asparagine obtained in step (c)    with a glycosidase;    (8) The process according to the above (6) or (7), wherein the    mixture of one or more sugar chain asparagines comprises:

and/or a compound having one or more deletions of sugar residues in theabove compound;(9) the process according to any one of the above (6) to (8), whereinthe fat-soluble protecting group is Fmoc group;(10) the process according to any one of the above (6) to (8), whereinthe step (a) is a step of introducing Fmoc group into the sugar chainaspargine contained in a mixture of one or more sugar chain asparagineshaving a sialic acid residue at a non-reducing terminal, and introducingbenzyl group into the sialic acid residue, to give a mixture of sugarchain asparagine derivatives;(11) a process for preparing a sugar chain, comprising the steps of:

-   (a) introducing a fat-soluble protecting group into a sugar chain    aspargine contained in a mixture of one or more sugar chain    asparagines, to give a mixture of sugar chain asparagine    derivatives;-   (b) subjecting the mixture of sugar chain asparagine derivatives or    a mixture obtainable by hydrolyzing a sugar chain asparagine    derivative contained in the mixture of sugar chain asparagine    derivatives to chromatography, to separate each of the sugar chain    asparagine derivatives therefrom;-   (c) removing the protecting group from the sugar chain asparagine    derivative separated in the step (b), to give a sugar chain    asparagine; and-   (d) removing an asparagine residue from the sugar chain asparagine    obtained in the step (c), to give the sugar chain;    (12) the process the above (11), further comprising the step of:-   (b′) hydrolyzing the sugar chain asparagine derivative separated in    step (b) with a glycosidase; and/or-   (c′) hydrolyzing the sugar chain asparagine obtained in step (c)    with a glycosidase; and/or-   (d′) hydrolyzing the sugar chain obtained in step (d) with a    glycosidase;    (13) the process according to the above (11) or (12), wherein the    mixture of one or more sugar chain asparagines comprises:

and/or a compound having one or more deletions of sugar residues in theabove compound;(14) the process according to any one of the above (11) to (13), whereinthe fat-soluble protecting group is Fmoc group;(15) the process according to any one of the above (11) to (13), whereinthe step (a) is a step of introducing Fmoc group into the sugar chainaspargine contained in a mixture of one or more sugar chain asparagineshaving a sialic acid residue at a non-reducing terminal, and introducingbenzyl group into the sialic acid residue, to give a mixture of sugarchain asparagine derivatives;(16) a sugar chain asparagine derivative having the general formula:

wherein R¹ and R², which may be identical or different, are H,

with proviso that a case where R¹ and R² are both

is excluded;(17) a sugar chain asparagine derivative having the general formula:

wherein one of R^(x) and R^(y) is

and the other is H,

(18) a sugar chain asparagine having the general formula:

wherein R³ and R⁴, which may be identical or different, are H,

with proviso that a case where R³ and R⁴ are both

is excluded; and(19) a sugar chain having the general formula:

wherein R⁵ and R⁶, which may be identical or different, are H,

with proviso that a case where R⁵ and R⁶ are both

or one of R⁵ or R⁶ is H and the other is

is excluded.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a group of structures of sugar chain asparagine derivativesobtainable according to the present invention.

FIG. 2 shows a group of structures of sugar chain asparagine derivativesobtainable according to the present invention.

FIG. 3 shows a group of structures of sugar chain asparagines obtainableaccording to the present invention.

FIG. 4 shows a group of structures of sugar chain asparagines obtainableaccording to the present invention.

FIG. 5 shows a group of structures of sugar chains obtainable accordingto the present invention.

FIG. 6 shows a group of structures of sugar chains obtainable accordingto the present invention.

FIG. 7 shows an example of a step in the process for preparing a sugarchain asparagine derivative according to the present invention.

FIG. 8 shows an example of a conversion step of a sugar chain asparaginederivative with various glycosidases.

FIG. 9 shows an example of a conversion step of a sugar chain asparaginederivative with various glycosidases.

FIG. 10 shows an example of a conversion step of a sugar chainasparagine derivative with various glycosidases.

FIG. 11 shows an example of a conversion step of a sugar chainasparagine derivative with various glycosidases.

FIG. 12 shows an example of a step of removing a protecting group (Fmocgroup) from a sugar chain asparagine derivative and an example of a stepof removing an asparagine residue from a sugar chain asparagine.

BEST MODE FOR CARRYING OUT THE INVENTION

One of the features of the process for preparing a sugar chainasparagine derivative of the present invention resides in that theprocess comprises introducing (binding) a fat-soluble protecting groupinto (to) a sugar chain asparagine derived from, for instance, anaturally occurring glycoprotein, preferably a sugar chain asparaginecontained in a mixture of sugar chain asparagines obtained fromasparagine type sugar chains, to give a mixture of sugar chainasparagine derivatives, and thereafter separating the mixture into eachof the sugar chain asparagine derivatives. The term “sugar chainasparagine” as used herein refers to a sugar chain having asparagine ina binding state. Also, the term “asparagine type sugar chain” refers toa group of sugar chains in each of which N-acetylglucosamine existing ina reducing terminal is bound by N-glycoside linkage to an acid aminogroup of asparagine (Asn) in a polypeptide of a protein, wherein thegroup of sugar chains have Man-β-(1-4)-GlcNAc-β-(1-4)-GlcNAc as a mothercore. The term “sugar chain asparagine derivative” refers to a sugarchain asparagine having a fat-soluble protecting group in a state ofbinding to an asparagine residue. Also, in the structural formula of thecompound, “AcHN” denotes acetamide group.

As mentioned above, the sugar chain derived from a naturally occurringglycoprotein is a mixture of sugar chains having random deletions ofsugar residues at non-reducing terminals. The present inventors havesurprisingly found that a fat-soluble protecting group is introducedinto a sugar chain derived from a naturally occurring glycoprotein,concretely a sugar chain asparagine contained in a mixture of sugarchain asparagines, so that a mixture of the resulting sugar chainasparagine derivatives into which the protecting groups are introducedcan be easily separated to individual sugar chain asparagine derivativesby means of a known chromatography. As a result, each of the sugar chainasparagine derivatives having a diversity of structures can be preparedin a large amount. For instance, the sugar chain asparagine derivativeshaving analogous structures, which had been conventionally difficult toseparate, such as compounds 2 and 6 shown in FIG. 1 and compounds 3 and7 shown in FIG. 2, can be separated, so that each of those compounds canbe prepared easily in a large amount. In addition, many kinds of thesugar chain asparagine derivatives can be synthesized more by, forinstance, removing the sugar residues from the resulting sugar chainasparagine derivative sequentially with glycosidases.

As described above, the individual sugar chain asparagine derivativescan be separated by introducing a fat-soluble protecting group into asugar chain asparagine to derivatize the sugar chain asparagine. This ispresumably due to the fact that a fat solubility of the overall sugarchain asparagine derivatives is increased by the introduction of thefat-soluble protecting group, so that an interaction with, for instance,a preferably employed reverse phase column is markedly improved, therebyresulting in even more sensitively separating individual sugar chainasparagine derivatives reflecting the difference in the sugar chainstructures. For instance, the fat solubility of Fmoc group, which is afat-soluble protecting group preferably used in the present invention,is very high. In other words, the fluorenyl backbone in Fmoc group has avery highly fat-soluble structure in which two benzene rings are boundto the 5-membered ring center. For instance, it is thought that a verystrong interaction with octadecyl group in ODS column is generated, theODS column being one of the reverse phase columns, whereby the sugarchain asparagine derivatives having analogous structures can beseparated.

Further, according to the present invention, there can be obtainedconveniently in a large amount various sugar chain asparagines byremoving the protecting group of the resulting sugar chain asparaginederivatives, and also various sugar chains by removing the asparagineresidues from the resulting sugar chain asparagines, as described below.

Concretely, the process for preparing a sugar chain asparaginederivative according to the present invention comprises the steps of:

-   (a) introducing a fat-soluble protecting group into the sugar chain    asparagine contained in a mixture of one or more sugar chain    asparagines, to give a mixture of sugar chain asparagine    derivatives; and-   (b) subjecting the mixture of sugar chain asparagine derivatives or    a mixture obtainable by hydrolyzing a sugar chain asparagine    derivative contained in the mixture of sugar chain asparagine    derivatives to chromatography, to separate each of the sugar chain    asparagine derivatives therefrom.

The mixture of one or more sugar chain asparagines usable in the step(a) is not particularly limited, as long as the mixture is a mixture ofone or more sugar chains each having asparagine in a binding state. Forinstance, the mixture may be a mixture of one or more sugar chains towhich one or more asparagines are bound. Among them, the mixture of oneor more sugar chains having asparagines bound at reducing terminals ispreferred from the viewpoint of facilitation of availability. The term“sugar chain” as used herein refers to a sugar chain in which two ormore monosaccharides are bound.

The mixture of the sugar chain asparagines can be obtained by a knownprocess, preferably by obtaining a mixture of glycoproteins and/orglycopeptides from naturally occurring raw materials, such as milk,bovine-derived fetuin or egg, and adding to the mixture an enzyme suchas protease, for instance, pronase (manufactured by Wako Pure ChemicalIndustries, Ltd.), Actinase-E (manufactured by Kaken Pharmaceutical Co.,Ltd.) or a general carboxypeptidase or aminopeptidase to carry out areaction under known reaction conditions to cleave a peptide moiety,thereby giving the mixture of the sugar chain asparagines as a reactionmixture after the reaction, or further removing the components otherthan the sugar chain asparagines from the reaction mixture by a knownprocess, such as purification process using various chromatographicmethods using gel filtration columns, ion exchange columns and the likeand a high-performance liquid chromatography (HPLC), thereby giving themixture of the sugar chain asparagines as a resulting product. From theviewpoint of the facilitation in the preparation, it is preferable thatthe above mixture is prepared by using a known egg-derived glycopeptide[Biochimica et Biophysica Acta 1335 (1997) p 23–32; crude SGP (a mixturecontaining proteins in egg yolk, an inorganic salt and the like, whereinthe glycopeptide is contained in an amount of from about 10 to about 80%by weight)].

Also, it is more preferable that the sugar chain asparagines containedin the mixture are hydrolyzed to previously cleave a part of sugarresidues thereof, from the viewpoint of efficiently obtaining the sugarchain asparagine derivatives having desired sugar chain structures. Thedegree of the cleavage is not particularly limited as long as thestructure is retained within the scope of the term “sugar chain” as usedherein. The mixture obtained as described above includes, for instance,a mixture containing a compound 24 shown in FIG. 3 and/or a compoundhaving one or more deletions of sugar residues in the above compound. Inthis case, the upper limit of deletions of sugar residues from thecompound is 9, from the viewpoint of retaining the structure of the“sugar chain” as used herein in the compound.

For instance, compounds 25 and 29 shown in FIG. 3 can be efficientlyobtained in a mixture of the sugar chain asparagines containing thecompound 24 (hereinafter referred to as a compound 24 mixture) bysubjecting the mixture to an acid hydrolysis in the manner as describedbelow, and furthermore the compounds 2 and 6 shown in FIG. 1, which arecorresponding sugar chain asparagine derivatives, can be obtainedefficiently.

For instance, an appropriate amount of an about 0.1 N aqueous acidicsolution is added to the compound 24 mixture to carry out a reaction,for instance, at 4° to 100° C. During the reaction, with monitoring theprogress of hydrolytic reaction by thin layer chromatography (forinstance, silica gel 60F254 (manufactured by Merck), ethylacetate:methanol:water=4:4:3 being used as an eluent), the reaction isstopped at a point where the compounds 25 and 29 can be obtained in thelargest amount. For instance, the reaction may be stopped after about 5to 10 hours under the condition of 25° C., or after about severalminutes under the condition of 100° C. Preferably, the reaction iscarried out at 70° C., the reaction is stopped 35 minutes after thebeginning of the reaction, and the mixture is cooled on ice rapidly. Thereaction can be stopped by neutralizing the reaction solution. Inaddition, the above acid is not particularly limited. For instance,there can be used inorganic acids and organic acids such as hydrochloricacid, sulfuric acid, nitric acid and trifluoroacetic acid, a cationicexchange resin, an insoluble solid reagent and the like.

Similarly, a compound 33 can be efficiently obtained from the compound24 in the above mixture. For instance, an appropriate amount of theabove aqueous acidic solution is added to the compound 24 mixture, thereaction is carried out preferably at 80° C., and the reaction isstopped at preferably 60 minutes after the beginning of the reaction.

The hydrolysis may also be carried out enzymatically. The enzyme used inthe reaction is preferably a glycosidase, and an enzyme of either endo-or exo-reaction forms can be used. For instance, when the compounds 25and 29 are obtained from the compound 24 in the same manner as describedabove, a sialic acid hydrolase having an activity of cleaving sialicacid at a terminal can be used. The enzyme is not particularly limited,and the enzyme may be any of commercially available enzymes, newlyisolated enzymes, enzymes generated by means of genetic engineering andthe like, as long as the enzyme has the above activity. The enzymaticreaction may be carried out in accordance with a known condition. Duringthe reaction, the progress is monitored by thin layer chromatography inthe same manner as described above, and the reaction may beappropriately stopped at a point where the compounds 25 and 29 areobtained in the largest amounts.

The fat-soluble protecting group is introduced into the sugar chainasparagine contained in the mixture of sugar chain asparagines using themixture obtained as described above. The protecting group is notparticularly limited, and there can be used, for instance, acarbonate-based or amide-based protecting group, such as Fmoc group,t-butyloxycarbonyl (Boc) group, benzyl group, allyl group,allyloxycarbonate group, or acetyl group. From the viewpoint that theresulting sugar chain asparagine derivative can be immediately used inthe synthesis of a desired glycopeptide, the above protecting group ispreferably Fmoc group, Boc group or the like, more preferably Fmocgroup. The Fmoc group is especially effective when there exists in thesugar chain a sugar, such as sialic acid, which is relative unstableunder acidic conditions. The introduction of the protecting group may becarried out according to a known process (for instance, ProtectingGroups in Organic Chemistry, John Wiley & Sons INC., New York 1991, ISBN0-471-62301-6).

For instance, when Fmoc group is used, an appropriate amount of acetoneis added to the mixture containing sugar chain asparagines,9-fluorenylmethyl-N-succinimidyl carbonate and sodium hydrogencarbonateare further added thereto and dissolved, and thereafter the resultingmixture is subjected to a binding reaction of Fmoc group to anasparagine residue at 25° C., whereby the Fmoc group can be introducedinto the asparagine residue of the above sugar chain asparagine.

According to the procedures described above, a mixture of the sugarchain asparagine derivatives into each of which a fat-soluble protectinggroup is introduced is obtained.

Next, in the step (b), the mixture of the sugar chain asparaginederivatives is subjected to a known chromatography, especially afractional chromatography, to separate each of the sugar chainasparagine derivatives therefrom. In this step, the mixture of sugarchain asparagine derivatives obtained in the above step (a) can bedirectly used. Alternatively, there may be used a mixture of the sugarchain asparagine derivatives obtained by further subjecting sugar chainasparagine derivatives contained in the above mixture to hydrolysis topreviously cleave a part of the sugar residues thereof, from theviewpoint of efficiently obtaining sugar chain asparagine derivativeshaving the desired sugar chain structures. The degree of the cleavage ofthe sugar residues is the same as that described above. In addition, thehydrolysis may be carried out in the same manner as described above.

For instance, when compounds 3 and 7 are obtained, the mixturecontaining compounds 2 and 6 is subjected to hydrolytic treatment with agalactosidase, instead of separating the compounds 3 and 7 from themixture, whereby the compounds 3 and 7 can be further easily separatedfrom the resulting mixture by HPLC, and each of the compounds can beobtained as an isolated product in a large amount.

The separation of each of sugar chain asparagine derivatives bychromatography can be carried out by appropriately using knownchromatographies, singly or in a combination of plural chromatographies.

For instance, the resulting mixture of sugar chain asparaginederivatives is purified by a gel filtration column chromatography, andthen purified by using HPLC. The column which can be used in HPLC ispreferably a reverse phase column, for instance, ODS, phenyl-based,nitrile-based, or anion exchange-based column, and concretely, a monoQcolumn manufactured by Pharmacia, Iatro-beads column manufactured byIatron can be utilized. The separation conditions and the like may beadjusted by referring to a known condition. According to the aboveprocedures, each of the desired sugar chain asparagine derivatives canbe obtained from the mixture of sugar chain asparagine derivatives. FIG.7 schematically shows an example of a step in the process for preparinga sugar chain asparagine derivative of the present invention.

Furthermore, the sugar chain asparagine derivative having a desiredsugar chain structure can be efficiently obtained by hydrolyzing thesugar chain asparagine derivatives separated in the step (b) [step(b′)]. For instance, in the stage of separating the sugar chainasparagine derivatives, the sugar chain asparagine derivatives can beroughly separated by limiting the kinds of the sugar chain asparaginederivatives contained in the mixture, and thereafter the sugar chainasparagine derivatives are subjected to hydrolysis, for instance,hydrolysis with a glycosidase, whereby the sugar chain asparaginederivatives having the desired sugar chain structures can be efficientlyobtained. Here, the hydrolysis can be carried out in the same manner asdescribed above. Especially, it is preferable that the hydrolysis iscarried out with a glycosidase of which cleavage mode of the sugarresidues is clear, from the viewpoint of more efficiently obtaining thesugar chain asparagine derivatives having the desired sugar chainstructures.

For instance, the conversion of the compounds 2 and 6 to the compounds 3and 7 by the removal of the galactose residues (FIG. 8) can beaccomplished by dissolving the compounds 2 and 6 in a buffer (forinstance, phosphate buffer, acetate buffer, Good's buffer or the like),and carrying out cleavage reaction of the galactose residues with agalactosidase in accordance with a known condition. The compounds 2 and6 may be a mixture of these or individually isolated compounds. It ispreferable that a commercially available known exo-form enzyme isutilized for the galactosidase used in this reaction. Also, the enzymemay be a newly isolated enzyme or an enzyme generated by geneticengineering, as long as the enzyme has a similar activity. Next, in thesame manner as described above, the reaction solution obtained after thereaction (a mixture of sugar chain asparagine derivatives of which sugarresidues are cleaved) may be subjected to chromatography to give each ofsugar chain asparagine derivatives. For instance, it is preferable thatthe separation is carried out by HPLC (ODS column, eluent being a 50 mMaqueous ammonium acetate:acetonitrile=82:15).

The conversion of the compounds 3 and 7 to the compounds 4 and 8 by theremoval of the N-acetylglucosamine residues (FIG. 8) can be accomplishedby dissolving the compounds 3 and 7 in a buffer (for instance, phosphatebuffer, acetate buffer, Good's buffer or the like), and carrying outcleavage reaction of the N-acetylglucosamine residues with anN-acetylglucosaminidase in accordance with a known condition. Also, anN-acetylhexosaminidase can be used. The compounds 3 and 7 may bemixtures of these or individually isolated compounds. It is preferablethat a commercially available known exo-form enzyme is utilized for eachenzyme used in this reaction. Also, the enzyme may be a newly isolatedenzyme or an enzyme generated by genetic engineering, as long as theenzyme has a similar activity. Next, in the same manner as describedabove, the reaction solution obtained after the reaction (a mixture ofsugar chain asparagine derivatives of which sugar residues are cleaved)is subjected to chromatography to give each of sugar chain asparaginederivatives. For instance, it is preferable that the separation iscarried out by HPLC (ODS column, eluent being a 50 mM aqueous ammoniumacetate:methanol=65:35 or a 50 mM aqueous ammoniumacetate:acetonitrile=82:15).

The conversion of the compounds 4 and 8 to the compounds 5 and 9 by theremoval of the mannose residues (FIG. 8) can be accomplished bydissolving the compounds 4 and 8 in a buffer (for instance, phosphatebuffer, acetate buffer, Good's buffer or the like), and carrying outcleavage reaction of the mannose residues with a mannosidase under aknown condition. The compounds 4 and 8 may be a mixture of these orindividually isolated compounds. It is preferable that a commerciallyavailable known exo-form enzyme is utilized for the mannosidase used inthis reaction. Also, the enzyme may be a newly isolated enzyme or anenzyme generated by genetic engineering, as long as the enzyme has asimilar activity. Next, in the same manner as described above, thereaction solution obtained after the reaction (a mixture of sugar chainasparagine derivatives of which sugar residues are cleaved) is subjectedto chromatography to give each of sugar chain asparagine derivatives.For instance, it is preferable that the separation is carried out byHPLC (ODS column, eluent: there can be used, for instance, a mixedsolution of a buffer such as an about 10 to about 200 mM ammoniumacetate and a water-soluble organic solvent with fat solubility such asacetonitrile, or ethanol, or methanol, or butanol, or propanol inappropriate amounts; when exemplified herein, it is preferable that theeluent is a 50 mM aqueous ammonium acetate:acetonitrile=82:18.).

The conversion of the compound 10 to the compound 11 by the removal ofthe galactose residues (FIG. 9) can be accomplished by dissolving acompound 10 in a buffer (for instance, phosphate buffer, acetate buffer,Good's buffer or the like), and carrying out cleavage reaction of thegalactose residues with a galactosidase in accordance with a knowncondition in the same manner as described above. It is preferable thatthe separation of the sugar chain asparagine derivatives from thereaction solution obtained after the reaction (a mixture of sugar chainasparagine derivatives of which sugar residues are cleaved) is carriedout by, for instance, HPLC (ODS column, eluent being a 50 mM aqueousammonium acetate:acetonitrile=85:15).

Also, by further hydrolyzing the compound 11 with an arbitraryglycosidase, the compound can be converted into various sugar chainasparagine derivatives (for instance, compounds 11, 12, 13 and thelike).

The conversion of the compound 11 to the compound 12 by the removal ofthe N-acetylglucosamine residues (FIG. 9) can be accomplished bydissolving the compound 11 in a buffer (for instance, phosphate buffer,acetate buffer, Good's buffer or the like), and carrying out cleavagereaction of the N-acetylglucosamine residues with anN-acetylglucosamidase under a known condition in the same manner asdescribed above. It is preferable that the separation of the sugar chainasparagine derivatives from the reaction solution obtained after thereaction (a mixture of sugar chain asparagine derivatives of which sugarresidues are cleaved) is carried out by, for instance, HPLC (ODS column,eluent being a 50 mM aqueous ammonium acetate:acetonitrile=85:18).

The conversion of the compound 12 to the compound 13 by the removal ofthe mannose residues (FIG. 9) can be accomplished by dissolving thecompound 12 in a buffer (for instance, phosphate buffer, acetate buffer,Good's buffer or the like), and carrying out cleavage reaction of themannose residues with a mannosidase in accordance with a known conditionin the same manner as described above. It is preferable that theseparation of each of sugar chain asparagine derivatives from thereaction solution obtained after the reaction (a mixture of sugar chainasparagine derivatives of which sugar residues are cleaved) is carriedout by, for instance, high-performance liquid column chromatography (ODScolumn, eluent being a 50 mM aqueous ammoniumacetate:acetonitrile=82:18).

The conversion of the compounds 3 and 7 to the compounds 14 and 19 (FIG.10) can be accomplished by dissolving the compounds 3 and 7 in a buffer(for instance, phosphate buffer, acetate buffer, Good's buffer or thelike), and carrying out cleavage reaction of the sialic acid residueswith a neuraminidase in accordance with a known condition. The compounds3 and 7 may be a mixture of these or individually isolated compounds. Itis preferable that a commercially available known exo-form enzyme isutilized for the sialic acid hydrolase used in this reaction. Also, theenzyme may be a newly isolated enzyme or an enzyme generated by geneticengineering, as long as the enzyme has a similar activity. Thereafter,in the same manner as described above, the reaction solution obtainedafter the reaction (a mixture of sugar chain asparagine derivatives ofwhich sugar residues are cleaved) is subjected to chromatography to giveeach of sugar chain asparagine derivatives. For instance, it ispreferable that the separation is carried out by high-performance liquidcolumn chromatography (ODS column, eluent being a 50 mM aqueous ammoniumacetate:acetonitrile=85:15).

The conversion of the compounds 14 and 19 to the compounds 15 and 20 bythe removal of the N-acetylglucosamine residues (FIG. 10) can beaccomplished by dissolving the compounds 14 and 19 in a buffer (forinstance, phosphate buffer, acetate buffer, Good's buffer or the like),and carrying out cleavage reaction of the N-acetylglucosamine residueswith an N-acetylglucosaminidase or the like in accordance with a knowncondition in the same manner as described above. It is preferable thatthe separation of each of sugar chain asparagine derivatives from thereaction solution obtained after the reaction (a mixture of sugar chainasparagine derivatives of which sugar residues are cleaved) is carriedout by, for instance, high-performance liquid column chromatography (ODScolumn, eluent being a 50 mM aqueous ammoniumacetate:acetonitrile=82:18).

The conversion of the compounds 15 and 20 to the compounds 16 and 21 bythe removal of the mannose residues (FIG. 10) can be accomplished bydissolving the compounds 15 and 20 in a buffer (for instance, phosphatebuffer, acetate buffer, Good's buffer or the like), and carrying outcleavage reaction of the mannose residues with a mannosidase inaccordance with a known condition in the same manner as described above.It is preferable that the separation of each of sugar chain asparaginederivatives from the reaction solution obtained after the reaction (amixture of sugar chain asparagine derivatives of which sugar residuesare cleaved) is carried out by, for instance, high-performance liquidcolumn chromatography (ODS column, eluent being a 50 mM aqueous ammoniumacetate:acetonitrile=82:18).

The conversion of the compounds 16 and 21 to the compounds 17 and 22 bythe removal of the galactose residues (FIG. 11) can be accomplished bydissolving the compounds 16 and 21 in a buffer (for instance, phosphatebuffer, acetate buffer, Good's buffer or the like), and carrying outcleavage reaction of the galactose residues with a galactosidase inaccordance with a known condition in the same manner as described above.It is preferable that the separation of each of sugar chain asparaginederivatives from the reaction solution obtained after the reaction (amixture of sugar chain asparagine derivatives of which sugar residuesare cleaved) is carried out by, for instance, high-performance liquidcolumn chromatography (ODS column, eluent being a 50 mM aqueous ammoniumacetate:acetonitrile=85:15).

The conversion of the compounds 17 and 22 to the compounds 18 and 23 bythe removal of the N-acetylglucosamine residues (FIG. 11) can beaccomplished by dissolving the compounds 17 and 22 in a buffer (forinstance, phosphate buffer, acetate buffer, Good's buffer or the like),and carrying out cleavage reaction of the N-acetylglucosamine residueswith an N-acetylglucosaminidase or the like in accordance with a knowncondition in the same manner as described above. It is preferable thatthe separation of each of sugar chain asparagine derivatives from thereaction solution obtained after the reaction (a mixture of sugar chainasparagine derivatives of which sugar residues are cleaved) is carriedout by, for instance, high-performance liquid column chromatography (ODScolumn, eluent being a 50 mM aqueous ammoniumacetate:acetonitrile=82:18).

As described above, each of the various sugar chain asparaginederivatives of which branching structures at the terminals of the sugarchains are not uniform, can be obtained as individual isolated compoundsby further hydrolyzing the derivatives with various glycosidases and thelike to remove the sugar residues at non-reducing terminals of the sugarchains after the obtainment of each of the sugar chain asparaginederivatives. Moreover, even a larger number of the kinds of the sugarchain asparagine derivatives can be prepared by changing the order orthe kind of hydrolysis with various glycosidases.

According to a conventional process, enormous amounts of time and costfor obtaining the sugar chain asparagine derivatives having very limitedsugar chain structures are required even on an analytical scale. On thecontrary, according to the present invention, about 1 gram of the sugarchain asparagine derivatives having desired sugar chain structures canbe prepared in an about 2-week period by using a conventional gelfiltration column, HPLC column, and at least three kinds of glycosidases(for instance, galactosidase, mannosidase, and N-acetylglucosamidase)without necessitating any particular devices or reagents.

In accordance with the procedures described above, when the protectinggroup, for instance, is Fmoc group, there can be efficiently obtained asugar chain asparagine derivative represented by the general formula:

wherein R¹ and R², which may be identical or different, are H,

with proviso that a case where R¹ and R² are both

is excluded.Concretely, the above sugar chain asparagine derivative includes, forinstance, each of the compounds shown in FIG. 1 and FIG. 2. Among them,the compound 1, the compounds 2 to 9, 11 to 23, 70 and 71 are compoundsprepared for the first time in the present invention. The presentinvention encompasses the above compounds.

In addition, as a preferred embodiment of the process for preparing asugar chain asparagine derivative of the present invention, there isprovided a process for preparing a sugar chain asparagine derivative inwhich the step (a) is a step of providing a mixture of one or more sugarchain asparagines each having a sialic acid residue at a non-reducingterminal, introducing Fmoc group into the sugar chain asparaginescontained in the above mixture, and introducing benzyl group into thesialic acid residue, to give a mixture of sugar chain asparaginederivatives. According to the above process, various forms of the sugarchain asparagine derivatives can be more efficiently obtained in a largeamount. For instance, the separation efficiency of the compounds 2 and 6shown in FIG. 1 can be increased, whereby the both compounds can beefficiently prepared. In other words, while there is a disadvantage inefficiently directly separating the compounds 2 and 6 definitely from amixture of the both compounds, in this embodiment, the both compoundscan be efficiently obtained from the mixture by introducing benzyl groupinto a sialic acid residue of the both compounds, and subjecting to theabove step (b) first of all as a mixture of the following compounds 76and 77:

Therefore, since the compounds 76 and 77 can be relatively easilyseparated, the compounds can be separated, and thereafter removing thebenzyl group from the compounds 76 and 77 by a process described belowenable compounds 2 and 6 to be efficiently separated and obtained fromthe mixture of the compounds.

Contrary to the somewhat difficulty in separation of the compounds 2 and6, it is thought that the reason why the separation of the compounds 76and 77, which are obtained by introducing benzyl group into a sialicacid residue of the compounds 2 and 6, can be easily carried out is thata highly fat-soluble benzyl group is introduced into carboxyl group ofthe sialic acid residue, so that a hydrophobic interaction with areverse phase column of HPLC (high-performance liquid chromatography) isfurther increased. Therefore, it is deduced that the interaction withthe reverse phase column preferably employed in the separation step (b)may be markedly improved, whereby consequently the separation of theboth compounds can be accomplished reflecting the difference in thesugar chain structure more sensitively.

The mixture of one or more sugar chain asparagines each having a sialicacid residue at a non-reducing terminal is not particularly limited, aslong as the mixture is a mixture of one or more sugar chain asparagineshaving the above structure. A mixture of one or more sugar chains eachhaving a sialic acid residue at a non-reducing terminal and also havingasparagine bound at a reducing terminal is preferred from the viewpointof easy availability. As the above mixture, a mixture containing thecompound 24 shown in FIG. 3 and/or a compound having one or moredeletions of sugar residues in the above compound is preferred.

It is preferable to carry out the above step (b′), from the viewpoint offurther efficiently obtaining a sugar chain asparagine derivative havingthe desired sugar chain structure.

In this embodiment, as the sugar chain asparagine derivatives, there areobtained those prepared by introducing benzyl group and Fmoc groupthereinto, and those prepared by introducing only Fmoc group thereinto.

The introduction of benzyl group into a sialic acid residue of the sugarchain asparagine may be carried out in accordance with a known process(for instance, see Protecting Groups in Organic Chemistry, John Wiley &Sons INC., New York 1991, ISBN 0-471-62301-6).

In accordance with the above procedures, for instance, there can beefficiently obtained a sugar chain asparagine derivative having benzylgroup and Fmoc group introduced thereinto, respectively, which isrepresented by the general formula:

wherein one of R^(x) and R^(y) is

and the other is H,

Concretely, the sugar chain asparagine derivative includes the abovecompounds 76 and 77, and the following compound 78:

The sugar chain asparagine derivative obtained according to thisembodiment can be directly used in solid phase synthesis of aglycopeptide. Since carboxyl group existing in a sialic acid residue ofthe sugar chain asparagine derivative is protected by benzyl group,there are some advantages that the desired sugar peptide can beefficiently obtained without causing side reactions involving thecarboxyl group in the solid phase synthesis of the glycopeptide, ascompared to the sugar chain asparagine derivative without introductionof benzyl group.

Also, the present invention provides a process for preparing a sugarchain asparagine capable of obtaining each of the various isolated sugarchain asparagines in a large amount. The above process furthercomprises, subsequent to the step of preparing a sugar chain asparaginederivative in accordance with the above process for preparing a sugarchain asparagine derivative, a step of removing the protecting groupfrom the resulting sugar chain asparagine derivative.

In other words, the process for preparing a sugar chain asparagineaccording to the present invention comprises the steps of:

-   (a) introducing a fat-soluble protecting group into a sugar chain    asparagine contained in a mixture of one or more sugar chain    asparagines, to give a mixture of sugar chain asparagine    derivatives;-   (b) subjecting the mixture of sugar chain asparagine derivatives or    a mixture obtainable by hydrolyzing a sugar chain asparagine    derivative contained in the mixture of sugar chain asparagine    derivatives to chromatography, to separate each of sugar chain    asparagine derivatives therefrom; and-   (c) removing the protecting group from the sugar chain asparagine    derivative separated in the step (b), to give a sugar chain    asparagine.

The steps (a) and (b) are the same as those in the above process forpreparing a sugar chain asparagine derivative, and the mixture of one ormore sugar chain asparagines used and the fat-soluble protecting groupused are also the same as those described above.

The removal of the protecting group from the sugar chain asparaginederivative in the step (c) can be carried out in accordance with a knownprocess (for instance, see Protecting Groups in Organic Chemistry, JohnWiley & Sons INC., New York 1991, ISBN 0-471-62301-6). For instance,when the protecting group is Fmoc group, the Fmoc group can be removedby adding morpholine to the sugar chain asparagine derivative inN,N-dimethylformamide (DMF) to carry out the reaction, as schematicallyshown in FIG. 12. On the other hand, Boc group can be removed by areaction with a weak acid. After the removal of the protecting group, asugar chain asparagine may be properly obtained by purifying a reactionmixture by a known process such as various chromatographies employing agel filtration column, an ion exchange column or the like or a processof separation by HPLC as desired.

In addition, similarly to the above process for preparing a sugar chainasparagine derivative, as a preferred embodiment of the process forpreparing a sugar chain asparagine of the present invention, there isprovided a process for preparing a sugar chain asparagine, in which thestep (a) is a step of providing a mixture of one or more sugar chainasparagines each having a sialic acid residue at a non-reducingterminal, introducing Fmoc group into the sugar chain asparaginescontained in the above mixture, and introducing benzyl group into thesialic acid residue, to give a mixture of sugar chain asparaginederivatives. In this embodiment, in the step (c), the removal of benzylgroup in addition to the removal of Fmoc group is carried out. Theremoval of benzyl group can be carried out in accordance with a knownprocess (for instance, see Protecting Groups in Organic Chemistry, JohnWiley & Sons INC., New York 1991, ISBN 0-471-62301-6).

Further, in the same manner as mentioned above, it is preferable tohydrolyze the sugar chain asparagine derivatives separated in the step(b) and/or hydrolyze the sugar chain asparagine obtained in the step(c), from the viewpoint of efficiently obtaining a sugar chainasparagine having a desired sugar chain structure. The hydrolysis can becarried out in the same manner as described above. It is more preferableto carry out the hydrolysis with a glycosidase [step (b′) and/or step(c′)], from the viewpoint of more efficiently obtaining a sugar chainasparagine having a desired sugar chain structure.

In accordance with the above procedures for instance, there can beefficiently obtained a sugar chain asparagine represented by the generalformula:

wherein R³ and R⁴, which may be identical or different, are H,

with proviso that a case where R³ and R⁴ are both

is excluded.Concretely, the sugar chain asparagine includes, for instance, each ofthe compounds shown in FIG. 3 and FIG. 4. Among them, the compounds 25to 32, 34 to 46, 72 and 73 are the compounds prepared for the first timein the present invention. The present invention encompasses the abovecompounds.

Further, the present invention provides a process for preparing a sugarchain capable of obtaining the various isolated sugar chains in a largeamount. The above process further comprises, subsequent to the step ofpreparing a sugar chain asparagine in accordance with the above processfor preparing a sugar chain asparagine, a step of removing an asparagineresidue from the resulting sugar chain asparagine.

In other words, the process for preparing a sugar chain of the presentinvention comprises the step of:

-   (a) introducing a fat-soluble protecting group into a sugar chain    asparagine contained in a mixture of one or more sugar chain    asparagines, to give a mixture of sugar chain asparagine    derivatives;-   (b) subjecting the mixture of sugar chain asparagine derivatives or    a mixture obtainable by hydrolyzing a sugar chain asparagine    derivative contained in the mixture of sugar chain asparagine    derivatives to chromatography, to separate each of sugar chain    asparagine derivatives therefrom;-   (c) removing the protecting group from the sugar chain asparagine    derivative separated in the step (b), to give a sugar chain    asparagine; and-   (d) removing an asparagine residue from the sugar chain asparagine    obtained in the step (c), to give the sugar chain.

The steps (a) to (c) are the same as those in the above process forpreparing a sugar chain asparagine, and the mixture of one or more sugarchain asparagines used and the fat-soluble protecting group used arealso the same as those mentioned above.

In addition, in the process for preparing a sugar chain of the presentinvention, as a preferred embodiment, there is provided a process forpreparing a sugar chain asparagine, in which the step (a) is a step ofproviding a mixture of one or more sugar chain asparagines each having asialic acid residue at a non-reducing terminal, introducing Fmoc groupinto the sugar chain asparagines contained in the above mixture, andintroducing benzyl group into the sialic acid residue to give a mixtureof sugar chain asparagine derivatives. In this embodiment, in the step(c), the removal of benzyl group in addition to the removal of Fmocgroup is carried out. The removal of benzyl group can be carried out inaccordance with the above process.

The removal of the asparagine residue from the sugar chain asparagine inthe step (d) can be carried out in accordance with a known process. Forinstance, the sugar chain asparagine is reacted with anhydrous hydrazineand then acetylated to remove the asparagine residue, whereby a sugarchain can be obtained, as schematically shown in FIG. 12. Also, a sugarchain can be also obtained by refluxing the sugar chain asparagine withheating in a basic aqueous solution and thereafter acetylating the sugarchain asparagine to remove the asparagine residue. After the removal ofthe asparagine residue, the sugar chain may be purified appropriately bya known process such as various chromatographies employing a gelfiltration column, an ion exchange column or the like, and a separationprocess by HPLC as desired.

Further, in the same manner as mentioned above, it is preferable tohydrolyze the sugar chain asparagine derivatives separated in the step(b) and/or hydrolyze the sugar chain asparagines obtained in the step(c), and/or hydrolyze the sugar chain obtained in the step (d), from theviewpoint of efficiently obtaining a sugar chain having a desired sugarchain structure. The hydrolysis may be carried out in the same manner asdescribed above. It is more preferable to carry out the hydrolysis witha glycosidase [the step (b′) and/or the step (c′) and/or the step (d′)],from the viewpoint of more efficiently obtaining a sugar chain having adesired sugar chain structure.

In accordance with the above procedures, for instance, there can beefficiently obtained a sugar chain represented by the general formula:

wherein R⁵ and R⁶, which may be identical or different, are H,

with proviso that a case where R⁵ and R⁶ are both

is excluded.Concretely, the sugar chain includes, for instance, each of thecompounds shown in FIG. 5 and FIG. 6. Among them, the compounds 48 to55, 57 to 69, 74 and 75 are the compounds prepared for the first time inthe present invention. The present invention encompasses the abovecompounds.

As described above, according to the present invention, the sugar chainasparagine derivative, the sugar chain asparagine and the sugar chain(hereinafter these three terms are collectively referred to as “sugarchain series” in some case) each having a desired sugar chain structurecan be prepared at a low cost, efficiently and in a large amount.

The above sugar chain series are very useful in the field of thedevelopment of pharmaceuticals and the like. An application example inthe development of the pharmaceuticals includes, for instance, asynthesis of cancer vaccine. It has been known that when the cell iscancerated, a sugar chain which is not initially existing in a body isexpressed. It has been also known that when the sugar chain issynthesized chemically and administered to an individual as a vaccine,the cancer proliferation is suppressed. Therefore, if a desired sugarchain series can be prepared according to the present invention, avaccine effective in the treatment of cancer can be synthesized. Also, anovel vaccine can be synthesized by derivatizing sugar chain seriesobtained according to the present invention by further bonding new sugarresidues using a combination of a chemical reaction and a sugartransferase reaction and the like.

In addition, for instance, erythropoietin (EPO), a glycoprotein, hasbeen used as a therapeutic agent for an anemia owing to itserythrocyte-proliferating ability, but it has been found that the EPOdoes not have any activity unless the EPO has a sugar chain boundthereto. As described above, some proteins exhibit their physiologicaleffects only under the binding of sugar chains. Therefore, for instance,a novel glycoprotein having a novel physiological effect can besynthesized by preparing a protein itself in a large amount in an E.coli expression system without binding ability of a sugar chain to theprotein, and subsequently introducing to the protein the sugar chainhaving a desired sugar chain structure, which is prepared according tothe present invention, to give a novel physiological activity to theprotein, or by introducing into a given protein the sugar chains havingvarious sugar chain structures, which are prepared according to thepresent invention.

In addition, a novel physiological activity can be imparted bysubstituting a sugar chain existing in a naturally occurringglycoprotein with a sugar chain prepared according to the presentinvention. A technique of substituting a sugar chain owned by aglycoprotein with a sugar chain obtained according to the presentinvention includes, for instance, a process described in P. Sears and C.H. Wong, Science, 2001, vol. 291, p. 2344–2350. In other words, theprocess includes a process of treating a glycoprotein with aβ-N-acetylglucosaminidase (Endo-H) to put the glycoprotein in a state inwhich only one N-acetylglucosamine residue is bound to the asparagineresidue on the surface of the protein, and subsequently binding adesired sugar chain in the sugar chain asparagine obtained according tothe present invention (for instance, each of the compounds shown in FIG.3 and FIG. 4) to the N-acetylglucosamine residue described above with aβ-N-acetylglucosaminidase (Endo-M). Alternatively, there can be carriedout a process of providing an N-acetylglucosamine-bound tRNA,synthesizing a glycoprotein having an N-acetylglucosamine residue byutilizing, for instance, an E. coli expression system, and thereafterintroducing a desired sugar chain in the sugar chain asparagine obtainedaccording to the present invention into the glycoprotein with Endo-M.

In addition, the problem encountered currently when a glycoprotein isutilized as a therapeutic agent includes a rapid metabolic rate of theglycoprotein administered. This is due to the fact that the glycoproteinis metabolized by the liver immediately after the removal of the sialicacid existing in the sugar chain terminal of the glycoprotein in aliving body. Therefore, it is necessary that the glycoprotein isadministered at a certain dose. In view of the above, if a sugar chainis prepared according to the present invention, wherein sialic acid isnewly introduced to a terminal of the sugar chain so as to be lessremoved, and the sugar chain is introduced into a target protein withEndo-M, the metabolic rate of the glycoprotein in a living body can becontrolled, so that the amount of the glycoprotein to be administeredcan be lowered.

The present invention will be more specifically described by means ofExamples, without intending to limit the present invention to theseExamples. The structural formulas and the numbers for each of thecompounds are shown in FIGS. 1 to 6. The data of ¹H-NMR were obtained bythe measurement with HOD at 4.8 ppm at 30° C. in Examples 1 to 7 andwith a signal of methyl group of acetone as an internal standard at2.225 ppm and HOD at 4.718 ppm at 30° C. in Examples 8 to 45. Also, thecompounds from which Fmoc groups had been removed were measured in theco-existence of a 50 mM ammonium hydrogencarbonate in a measurementsolvent.

EXAMPLE 1 Synthesis of Compound 24

In 100 ml of a tris-hydrochloric acid-calcium chloride buffer (TRIZMABASE 0.05 mol/l, calcium chloride 0.01 mol/l, pH 7.5) was dissolved 2.6g of an egg-derived crude SGP (sialyl glycopeptide). Fifty-eightmilligrams (772 μmol) of sodium azide and 526 mg of Actinase-E(manufactured by Kaken Pharmaceutical Co., Ltd.) were added to thissolution, and the mixture was allowed to stand at 37° C. After 65 hours,263 mg of Actinase-E was added again, and the mixture was allowed tostand at 37° C. for additional 24 hours. This solution was lyophilized,and thereafter the residue was purified twice by gel filtration columnchromatography (Sephadex G-25, 2.5φ×1 m, eluent: water, flow rate: 1.0ml/min), to give 1.3 g (555 μmol) of a desired compound 24 shown in FIG.3. The structure of the sugar chain contained in SGP is shown asfollows.

In addition, the physical data for the resulting compound 24 are asfollows.

¹H-NMR (D₂O, 30° C.) 5.15 (1H, s, Man4-H₁), 5.06 (1H, d, GlcNAc1-H₁),4.95 (1H, s, Man4′-H₁), 4.82 (1H, s, Man3-H₁), 4.69 (1H, d, GlcNAc2-H₁),4.67 (2H, d, GlcNAc5,5′-H₁), 4.53 (2H, d, Gal6,6′-H₁), 4.34 (1H, d,Man3-H₂), 4.27 (1H, d, Man4′-H₂), 4.19 (1H, d, Man4-H₂) 3.03 (1H, dd,Asn-βCH), 3.00 (1H, dd, Asn-βCH), 2.76 (2H, dd, NeuAc7,7′-H_(3eq)), 2.15(18H, s×6, —Ac), 1.79 (2H, dd, NeuAc7,7′-H_(3ax))

EXAMPLE 2 Synthesis of Compounds 1, 2, 6 and 10

The compound 24 (609 mg, 261 μmol) obtained in Example 1 was dissolvedin 20.7 ml of water, and 13.8 ml of 0.1 N hydrochloric acid was addedthereto. Immediately after heating this solution at 70° C. for 35minutes, the solution was cooled on ice, and a saturated aqueous sodiumhydrogencarbonate was added thereto to adjust its pH to 7. The solutionwas lyophilized, and thereafter the residue was purified by gelfiltration column chromatography (Sephadex G-25, 2.5φ×1 m, eluent:water, flow rate: 1.0 ml/min), to give 534 mg of a mixture of a compound24, compounds 25 and 29, and a compound 33 each shown in FIG. 3. Thesefour components were proceeded to the next step without being isolatedfrom each other.

The physical data for the resulting sugar chain mixture are as follows.

¹H-NMR (D₂O, 30° C.) 5.13 (s, Man4-H₁), 5.12 (s, Man4-H₁), 5.01 (d,GlcNAc1-H₁), 4.94 (s, Man4′-H₁), 4.93 (s, Man4′-H₁), 4.82 (s, Man3-H₁),4.60 (d, GlcNAc2-H₁), 4.58 (d, GlcNAc5,5′-H₁), 4.47 (dd, Gal6,6′-H₁),4.44 (d, Gal6,6′-H₁), 4.24 (d, Man3-H₂), 4.19 (d, Man4′-H₂), 4.11 (d,Man4-H₂), 2.97 (bdd, Asn-βCH), 2.72 (dd, NeuAc7-H_(3eq),NeuAc7-H_(3eq)), 2.64 (bdd, Asn-βCH), 2.15 (s×5, —Ac), 1.79 (dd,NeuAc7-H_(3ax), NeuAc7′-H_(3ax))

Four-hundred and twenty-nine milligrams of the mixture of the resultingsugar chain was dissolved in 16.3 ml of acetone and 11.2 ml of water. Tothis solution were added 9-fluorenyl methyl-N-succinimidyl carbonate(155.7 mg, 461.7 μmol) and sodium hydrogencarbonate (80.4 mg, 957 μmol),and the mixture was stirred at room temperature for 2 hours. Thissolution was applied to an evaporator to remove acetone, and theremaining solution was purified by gel filtration column chromatography(Sephadex G-25, 2.5φ×1 m, eluent: water, flow rate: 1.0 ml/min), to give309 mg of a mixture of a compound 1, compounds 2 and 6, and a compound10 each shown in FIG. 1. This mixture was purified by HPLC (ODS column,eluent: 50 mM aqueous ammonium acetate:methanol=65:35, 2.0φ×25 cm, flowrate: 3 ml/min). As a result, the compound 1 was eluted after 51minutes, a mixture of the compounds 2 and 6 was eluted after 67 minutes,and the compound 10 was eluted after 93 minutes. Each of the fractionswere collected and lyophilized, and thereafter desalted by gelfiltration column chromatography (Sephadex G-25, 2.5φ×30 cm, eluent:water, flow rate: 1.0 ml/min), thereby giving 150 mg of a desiredmixture of the compounds 2 and 6.

The physical data for the resulting compound 1 are as follows.

¹H-NMR (D₂O, 30° C.) 7.99 (2H, d, Fmoc), 7.79 (2H, d, Fmoc), 7.55 (4H,m, Fmoc), 5.15 (1H, s, Man4-H₁), 5.06 (1H, d, GlcNAc1-H₁), 4.95 (1H, s,Man4′-H₁), 4.82 (1H, s, Man3-H₁), 4.69 (1H, d, GlcNAc2-H₁), 4.67 (2H, d,GlcNAc5,5′-H₁), 4.53 (2H, d, Gal6,6′-H₁), 4.34 (1H, d, Man3-H₂), 4.27(1H, d, Man4′-H₂), 4.19 (1H, d, Man4-H₂), 3.03 (1H, bdd, Asn-βCH), 3.00(1H, bdd, Asn-βCH), 2.76 (2H, dd, NeuAc7,7′-H_(3eq)), 2.15 (18H, s×6,—Ac), 1.79 (2H, dd, NeuAc7,7′-H_(3ax)); HRMS Calcd forC₁₀₃H₁₅₄N₈NaO₆₆[M+Na⁺] 2581.8838, found 2581.8821.

The physical data for the resulting mixture of the compounds 2 and 6 areas follows.

¹H-NMR (D₂O, 30° C.) 7.99 (d, Fmoc), 7.79 (d, Fmoc), 7.55 (m, Fmoc),5.14 (s, Man4-H₁), 5.12 (s, Man4-H), 5.00 (d, GlcNAc1-H₁), 4.94 (s,Man4′-H₁), 4.93 (s, Man4′-H₁), 4.82 (s, Man3-H₁), 4.60 (d, GlcNAc2-H₁),4.58 (d, GlcNAc5,5′-H₁), 4.46 (dd, Gal6,6′-H₁), 4.44 (d, Gal6,6′-H₁),4.24 (d, Man3-H₂), 4.19 (d, Man4′-H₂), 4.11 (d, Man4-H₂), 2.97 (bdd,Asn-βCH), 2.72 (dd, NeuAc7-H_(3eq), NeuAc7-H_(3eq)), 2.64 (bdd,Asn-βCH), 2.15 (s×5, —Ac), 1.79 (dd, NeuAc7-H_(3ax), NeuAc7′-H_(3ax))

The physical data for the resulting compound 10 are as follows.

¹H-NMR (D₂O, 30° C.) 7.99 (2H, d, Fmoc), 7.79 (2H, d, Fmoc), 7.55 (4H,m, Fmoc), 5.12 (1H, s, Man4-H₁), 5.06 (1H, d, GlcNAc1-H₁), 4.93 (1H, s,Man4′-H₁), 4.82 (1H, s, Man3-H₁), 4.69 (1H, d, GlcNAc2-H₁), 4.67 (2H, d,GlcNAc5,5′-H₁), 4.53 (2H, d, Gal6,6′-H₁), 4.34 (1H, d, Man3-H₂), 4.27(1H, d, Man4′-H₂), 4.19 (1H, d, Man4-H₂), 3.03 (1H, bdd, Asn-βCH), 3.00(1H, bdd, Asn-βCH), 2.15 (12H, s×4, —Ac); HRMS Calcd forC₈₁H₁₂₀N₆NaO₅₀[M+Na⁺] 1999.6930, found 1999.6939.

EXAMPLE 3 Synthesis of Compounds 3 and 7

The mixture (224 mg, 97 μmol) of the compounds 2 and 6 obtained inExample 2 and 24 mg of bovine serum albumin were dissolved in 22 ml ofHEPES buffer (50 mM, pH 6.0), and Diplococcus pneumoniae-derivedβ-galactosidase (1.35 U) was added thereto. This solution was allowed tostand at 37° C. for 15 hours, and thereafter lyophilized. The residuewas purified by HPLC (ODS column, 2.0φ×25 cm, eluent: 50 mM aqueousammonium acetate:acetonitrile=85:15, flow rate: 3 ml/min), and acompound 3 shown in FIG. 2 was eluted after 129 minutes, and a compound7 was eluted after 134 minutes. Each of the fractions was collected andlyophilized. Subsequently, the fraction was desalted by HPLC (ODScolumn, 2.0φ×25 cm, eluent:water for a first 15 minutes, and applied toa gradient of water:acetonitrile of from 10:0 to 85:15 (volume ratio)for a period of from 16 to 30 minutes, and then to a gradient ofwater:acetonitrile from 85:15 to 80:20 for a period of from 31 to 45minutes; flow rate: 3.0 ml/min), to give a desired compound 3 in anamount of 81 mg and a compound 7 in an amount of 75 mg.

The physical data for the resulting compound 3 are as follows.

¹H-NMR (D₂O, 30° C.) 7.99 (2H, d, Fmoc), 7.79 (2H, d, Fmoc), 7.55 (4H,m, Fmoc), 5.15 (1H, S, Man4-H₁), 5.06 (1H, d, GlcNAc1-H₁), 4.95 (1H, s,Man4′-H₁), 4.82 (1H, s, Man3-H₁), 4.69 (1H, d, GlcNAc2-H₁), 4.67 (2H, d,GlcNAc5,5′-H₁), 4.53 (1H, d, Gal6′-H₁), 4.34 (1H, d, Man3-H₂), 4.27 (1H,d, Man4′-H₂), 4.19 (1H, d, Man4-H₂), 2.97 (1H, bdd, Asn-βCH), 2.76 (1H,dd, NeuAc7′-H_(3eq)), 2.61 (1H, bdd, Asn-βCH), 2.15 (1SH, s×5, —Ac),1.79 (1H, dd, NeuAc7′-H_(3ax)); HRMS Calcd for C₈₆H₁₂₇N₇NaO₅₃[M+Na⁺]2128.7356, found 2128.7363.

The physical data for the resulting compound 7 are as follows.

¹H-NMR(D₂O, 30° C.) 7.99 (2H, d, Fmoc), 7.79 (2H, d, Fmoc), 7.55 (4H, m,Fmoc), 5.15 (1H, S, Man4-H₁), 5.06 (1H, d, GlcNAc1-H₁), 4.95 (1H, s,Man4′-H₁), 4.82 (1H, s, Man3-H₁), 4.69 (1H, d, GlcNAc2-H₁), 4.67 (2H, d,GlcNAc5,5′-H₁), 4.53 (1H, d, Gal6-H₁), 4.34 (1H, d, Man3-H₂), 4.27 (1H,d, Man4′-H₂) 4.19 (1H, d, Man4-H₂), 2.97 (1H, bdd, Asn-βCH), 2.76 (1H,dd, NeuAc7-H_(3eq)), 2.60 (1H, b dd, Asn-βCH), 2.15 (15H, s×5, —Ac),1.79 (1H, dd, NeuAc7-H_(3ax)); HRMS Calcd for C₈₆H₁₂₅N₇Na₃O₅₃[M+Na⁺]2172.6995, found 2172.7084.

EXAMPLE 4 Synthesis of Compounds 4 and 8

A mixture (90 mg, 47.3 μmol) of the compounds 3 and 7 obtained inExample 3 was dissolved in 8.1 ml of HEPES buffer (50 mM, pH 6.0)together with 8 mg of bovine serum albumin without separating thecompounds from each other, and 2.88 U of a bovine kidney-derivedβ-glucosaminidase (manufactured by Sigma-Aldrich Corporation, frombovine kidney) was added thereto. This solution was allowed to stand at37° C. for 18 hours, and thereafter lyophilized. The residue waspurified by HPLC (ODS column, 2.0φ×25 cm, eluent: 50 mM aqueous ammoniumacetate:methanol=65:35, flow rate: 3 ml/min), and a compound 4 shown inFIG. 2 was eluted after 117 minutes, and a compound 8 was eluted after127 minutes. Each of the fractions was collected and lyophilized.Subsequently, the fraction was desalted by HPLC (ODS column, 2.0φ×25 cm,eluent: water for a first 15 minutes, and applied to a gradient ofwater:acetonitrile of from 10:0 to 85:15 for a period of from 16 to 30minutes, and then to a gradient of water:acetonitrile of from 85:15 to80:20 for a period of from 31 to 45 minutes; flow rate: 3.0 ml/min), togive a desired compound 4 in an amount of 40 mg and a compound 8 in anamount of 37 mg.

The physical data for the resulting compound 4 are as follows.

¹H-NMR (D₂O, 30° C.) 8.01 (2H, d, Fmoc), 7.80 (2H, d, Fmoc), 7.56 (4H,m, Fmoc), 5.22 (1H, s, Man4-H₁), 5.08 (1H, d, GlcNAc1-H₁), 4.94 (1H, s,Man4′-H₁), 4.84 (1H, s, Man3-H₁), 4.69 (1H, d, GlcNAc2-H₁), 4.67 (1H, d,GlcNAc5-H₁), 4.55 (1H, d, Gal6-H₁), 4.33 (1H, dd, Man3-H₂), 4.20 (1H,dd, Man4-H₂), 4.15 (1H, dd, Man4′-H₂), 2.97 (1H, bdd, Asn-βCH), 2.76(2H, dd, NeuAc7,7′-H_(3e,q)), 2.62 (1H, bdd, Asn-βCH), 2.15 (12H, s×4,—Ac), 1.79 (2H, dd, NeuAc7, 7′-H_(3ax)); HRMS Calcd forC₇₈H₁₁₄N₆NaO₄₈[M+Na⁺] 1925.6562, found 1925.6539.

The physical data for the resulting compound 8 are as follows.

¹H-NMR (D₂O, 30° C.) 7.99 (2H ,d, Fmoc), 7.79 (2H, d, Fmoc), 7.55 (4H,m, Fmoc), 5.15 (1H, S, Man4-H₁), 5.06 (1H, d, GlcNAc1-H₁), 4.95 (1H, s,Man4′-H₁), 4.82 (1H, s, Man3-H₁), 4.69 (1H, d, GlcNAc2-H₁), 4.67 (2H, d,GlcNAc5,5′-H₁), 4.53 (2H, d, Gal6,6′-H₁), 4.34 (1H, d, Man3-H₂), 4.27(1H, d, Man4′-H₂), 2.97 (1H, bdd, Asn-βCH₂), 2.76 (1H, dd,NeuAc7′-H_(3eq)), 2.61 (1H, bdd, Asn-βCH₂), 2.15 (12H, s×4, —Ac), 1.79(1H, dd, NeuAc7′-H_(3ax)); HRMS Calcd for C₇₈H₁₁₄N₆NaO₄₈[M+Na⁺]1925.6562, found 1925.6533.

EXAMPLE 5 Synthesis of Compound 5

The compound 4 (30 mg, 473 μmol) obtained in Example 4 and 3 mg ofbovine serum albumin were dissolved in 6 ml of HEPES buffer (50 mM, pH6.0), and 10 U of Jack Beans-derived a-mannosidase was added thereto.This solution was allowed to stand at 37° C. for 21 hours, and thenlyophilized. Subsequently, the residue was purified by HPLC (ODS column,2.0φ×25 cm, eluent:water for a first 15 minutes, and applied to agradient of water:acetonitrile of from 10:0 to 85:15 for a period offrom 16 to 30 minutes, and then to a gradient of water:acetonitrile offrom 85:15 to 80:20 for a period of from 31 to 45 minutes; flow rate:3.0 ml/min), to give 20 mg of a desired compound 5 shown in FIG. 1.

The physical data for the resulting compound 5 are as follows.

¹H-NMR (D₂O, 30° C.) 8.01 (2H, d, Fmoc), 7.80 (2H, d, Fmoc), 7.56 (4H,m, Fmoc), 5.00 (1H, d, GlcNAc1-H₁), 4.95 (1H, s, Man4′-H₁), 4.84 (1H, s,Man3-H₁), 4.67 (1H, d, GlcNAc2-H₁), 4.56 (1H, d, GlcNAc5-H₁), 4.44 (1H,d, Gal6-H₁), 4.11 (1H, dd, Man4′-H₂), 4.07 (1H, dd, Man3-H₂), 2.97 (1H,bdd, Asn-βCH), 2.76 (1H, dd, NeuAc7′-H_(3e,q)), 2.62 (1H, bdd, Asn-βCH),2.15 (12H, s×4, —Ac), 1.79 (2H, dd, NeuAc7′-H_(3ax)); HRMS Calcd forC₇₂H₁₀₄N₆NaO₄₃[M+Na⁺] 1763.6034, found 1763.6074.

EXAMPLE 6 Synthesis of Compound 9

The compound 8 (40 mg, 630 μmol) obtained in Example 4 and 5 mg ofbovine serum albumin were dissolved in 7.8 ml of HEPES buffer (50 mM, pH6.0), and 38 U of a Jack Beans-derived α-mannosidase was added thereto.This solution was allowed to stand at 37° C. for 63 hours, and thenlyophilized. Subsequently, the residue was purified by HPLC (ODS column,2.0φ×25 cm, eluent:water for a first 15 minutes, and applied to agradient of water:acetonitrile of from 10:0 to 85:15 for a period offrom 16 to 30 minutes, and then to a gradient of water:acetonitrile offrom 85:15 to 80:20 for a period of from 31 to 45 minutes; flow rate:3.0 ml/min), to give 30 mg of a desired compound 9.

The physical data for the resulting compound 9 are as follows.

¹H-NMR (D₂O, 30° C.) 8.01 (2H, d, Fmoc), 7.80 (2H, d, Fmoc), 7.56 (4H,m, Fmoc), 5.23 (1H, s, Man4-H₁), 5.08 (1H, d, GlcNAc1-H₁), 4.53 (1H, d,Gal6-H₁), 4.32 (1H, dd, Man3-H₂), 4.28 (1H, dd, Man4-H₂), 2.81 (1H, bdd,Asn-βCH), 2.76 (1H, dd, NeuAc7-H_(3eq)), 2.59 (1H, bdd, Asn-βCH), 2.13(12H, s×4, —Ac), 1.80 (1H, dd, NeuAc7H_(3ax)); HRMS Calcd forC₇₂H₁₀₄N₆NaO₄₃[M+Na⁺] 1763.6034, found 1763.6041.

EXAMPLE 7 Deprotection of Fmoc Group (Synthesis of Compound 33)

The compound 10 (10.5 mg, 5.27 μmol) obtained in Example 2 was dissolvedin 1.4 ml of a 50% morpholine/N,N-dimethylformamide solution, and thesolution was reacted for 2 hours under argon atmosphere at roomtemperature. Three milliliters of toluene was added to this solution,and the mixture was applied to an evaporator at 35° C. The procedureswere repeated three times to remove the reaction solvent. The residuewas purified by gel filtration column chromatography (Sephadex G-25,2.5φ×30 cm, eluent: water, flow rate: 1.0 ml/min), to give 7 mg of adesired compound 33 shown in FIG. 3 (yield: 76%). The structure of theresulting compound was confirmed from the finding that its ¹H-NMRspectrum was identical to that of the resulting compound 33 from Example2.

The physical data for the resulting compound 33 are as follows.

¹H-NMR (30° C.) δ5.12 (s, 1H, Man4-H-1), 5.07 (d, 1H, J=9.7 Hz,GlcNAc1-H-1), 4.92 (s, 1H, Man4′-H-1), 4.76 (s, 1H, Man3-H-1), 4.62 (d,1H, J=8.0 Hz, GlcNAc2-H-1), 4.58 (d, 2H, J=7.8 Hz, GlcNAc5,5′-H-1), 4.47(d, 2H, J=7.9 Hz, Gal6,6′-H-1), 4.24 (bd, 1H, Man3-H-2), 4.19 (bdd, 1H,J=3.2 Hz, 1.4 Hz, Man4′-H-2), 4.12 (bdd, 1H, J=3.2 Hz, 1.4 Hz,Man4-H-2), 2.93 (dd, 1H, J=4.5 Hz, 17.0 Hz, Asn-βCH), 2.93 (dd, 1H,J=6.8 Hz, 17.0 Hz, Asn-βCH), 2.08 (s, 3H, Ac), 2.05 (s, 6H, Ac×2), 2.01(s, 3H, Ac)

EXAMPLE 8 Synthesis of Compound 14

The compound 3 (28 mg, 21.3 μmol) and 1.0 mg of bovine serum albuminwere dissolved in HEPES buffer (50 mM, pH 5.0, 454 μL), andneuraminidase (manufactured by Sigma-Aldrich Corporation, from ViblioCholerae, 198 mU) was added thereto. This solution was allowed to standat 37° C. for 20 hours, and thereafter the termination of the reactionwas confirmed by HPLC analysis. The reaction solution was purified byHPLC (YMC Packed Column D-ODS-5 S-5 120A ODS No. 2020178, 20×250 mm,eluent: 50 mM aqueous ammonium acetate:acetonitrile=80:20, flow rate: 4mL/min). Further, the residue was desalted on ODS column (Cosmosil75C₁₈-OPN, 15×100 mm, eluted first with 50 mL of H₂O and then with 25%acetonitrile), to give a desired compound 14 (17 mg, yield: 70%). Thephysical data for the resulting compound are as follows.

¹H-NMR (30° C.) δ7.91 (d, 2H, J=7.5 Hz, Fmoc), 7.71 (d, 2H, J=7.5 Hz,Fmoc), 7.51 (dd, 2H, J=7.5 Hz, Fmoc), 7.43 (dd, 2H, J=7.5 Hz, Fmoc),5.12 (s, 1H, Man4-H-1), 4.99 (d, 1H, J=9.5 Hz, GlcNAc1-H-1), 4.92 (s,1H, Man4′-H-1), 4.76 (s, 1H, Man3-H-1), 4.58 (d, 1H, J=8.0 Hz,GlcNAc2-H-1), 4.55 (d, 1H, J=8.4 Hz, GlcNAc5′-H-1) 4.47 (d, 1H, J=7.8Hz, Gal6′-H-1), 4.34 (t, 1H, Fmoc), 4.24 (bd, 1H, J=1.9 Hz Man3-H-2),4.18 (bdd, 1H, J=1.4 Hz, 3.3 Hz, Man4-H-2), 4.11 (bdd, 1H, J=1.4 Hz, 3.5Hz, Man4′-H-2), 2.72 (bdd, 1H, J=3.0 Hz, 15.7 Hz, Asn-βCH), 2.52 (bdd,1H, J=8.7 Hz, 15.7 Hz, Asn-βCH), 2.06, 2.05, 2.04, 1.89 (each s, each3H, Ac); HRMS Calcd for C₇₅H₁₁₀N₆NaO₄₅[M+Na⁺] 1837.6402, found1837.6471.

EXAMPLE 9 Synthesis of Compound 19

The compound 7 (20 mg, 9.4 μmol) and 1.6 mg of bovine serum albumin weredissolved in HEPES buffer (50 mM, pH 5.0, 323 μL), and neuraminidase(manufactured by Sigma-Aldrich Corporation, from Viblio Cholerae, 141mU) was added thereto. This solution was allowed to stand at 37° C. for18 hours, and thereafter the termination of the reaction was confirmedby HPLC analysis. Subsequently, the reaction solution was purified byHPLC (YMC Packed Column D-ODS-5 S-5 120A ODS No. 2020178, 20×250 mm,eluent: 50 mM aqueous ammonium acetate:acetonitrile=80:20, flow rate: 4mL/min). Further, the residue was desalted on an ODS column (Cosmosil75C₁₈-OPN, 15×100 mm, eluted first with 50 mL of H₂O and then with 25%acetonitrile), to give a desired compound 19 (13 mg, yield: 76%). Thestructure of the resulting compound was confirmed from the finding thatits ¹H-NMR was identical to that of the preparation.

EXAMPLE 10 Synthesis of Compound 15

The compound 4 (45 mg, 24 μmol) and 1.7 mg of bovine serum albumin weredissolved in HEPES buffer (50 mM, pH 5.0, 820 μL), and neuraminidase(manufactured by Sigma-Aldrich Corporation, from Viblio Cholerae, 134mU) was added thereto. This solution was allowed to stand at 37° C. for14 hours, and thereafter the termination of the reaction was confirmedby HPLC analysis. Subsequently, the reaction solution was purified byHPLC (YMC Packed Column D-ODS-5 S-5 120A ODS No. 2020178, 20×250 mm,eluent: 50 mM aqueous ammonium acetate:acetonitrile=80:20, flow rate: 4mL/min). Further, the residue was desalted on an ODS column (Cosmosil75C₁₈-OPN, 15×100 mm, eluted first with 50 mL of H₂O and then with 25%acetonitrile), to give a desired compound 15 (28 mg, yield: 74%). Thephysical data for the resulting compound are as follows.

¹H-NMR (30° C.) δ7.92 (d, 2H, J=7.5 Hz, Fmoc), 7.71 (d, 2H, J=7.5 Hz,Fmoc), 7.51 (dd, 2H, J=7.5 Hz, Fmoc), 7.44 (dd, 2H, J=7.5 Hz, Fmoc),5.10 (s, 1H, Man4-H-1), 4.99 (d, 1H, J=9.5 Hz, GlcNAc1-H-1), 4.92 (s,1H, Man4′-H-1), 4.76 (s, 1H, Man3-H-1), 4.58 (d, 2H, GlcNAc2,5′-H-1),4.47 (d, 1H, J=8.0 Hz, Gal6′-H-1), 4.35 (t, 1H, Fmoc), 4.24 (bd, 1H,J=1.9 Hz, Man3-H-2), 4.11 (bs, 1H, Man4′-H-2), 4.07 (bs, 1H, Man4-H-2),2.72 (bd, 1H, J=15.5 Hz, Asn-βCH), 2.52 (bdd, 1H, J=8.7 Hz, 15.5 Hz,Asn-βCH), 2.06, 2.04, 1.89 (each s, each 3H, Ac); HRMS Calcd forC₆₇H₉₇N₅NaO₄₀[M+Na⁺] 1634.5608, found 1634.5564.

EXAMPLE 11 Synthesis of Compound 70

The compound 15 (11 mg, 6.8 μmol) and 1.5 mg of bovine serum albuminwere dissolved in HEPES buffer (50 mM, pH 5.0, 269 μL), andβ-galactosidase (manufactured by Seikagaku Corporation, from Jack Beans,11 μL, 275 mU) was added thereto. This solution was allowed to stand at37° C. for 14 hours, and thereafter the termination of the reaction wasconfirmed by HPLC analysis. The reaction solution was purified by HPLC(YMC Packed Column D-ODS-5 S-5 120A ODS No. 2020178, 20×250 mm, eluent:50 mM aqueous ammonium acetate:acetonitrile=80:20, flow rate: 4 mL/min).Further, the residue was desalted on an ODS column (Cosmosil 75C₁₈-OPN,15×100 mm, eluted first with 50 mL of H₂O and then with 25%acetonitrile), to give a desired compound 70 (6.3 mg, yield: 64%). Thephysical data for the resulting compound are as follows.

¹H-NMR (30° C.) δ7.91 (d, 2H, J=7.5 Hz, Fmoc), 7.70 (d, 2H, J=7.5 Hz,Fmoc), 7.50 (dd, 2H, J=7.5 Hz, Fmoc), 7.43 (dd, 2H, J=7.5 Hz, Fmoc),5.10 (s, 1H, Man4-H-1), 4.99 (d, 1H, J=9.5 Hz, GlcNAc1-H-1), 4.91 (s,1H, Man4′-H-1), 4.76 (s, 1H, Man3-H-1), 4.55 (d, 2H, GlcNAc2,5′-H-1),4.32 (t, 1H, Fmoc), 4.24 (bs, 1H, Man3-H-2), 4.10 (bs, 1H, Man4-H-2),4.06 (bs, 1H, J=1.3 Hz, Man4′-H-2), 2.72 (bd, 1H, J=14.0 Hz, Asn-βCH),2.52 (bdd, 1H, J=9.5 Hz, 14.8 Hz, Asn-βCH), 2.06, 2.05, 1.89 (each s,each 3H, Ac); MS(Fab) Calcd for C₆₁H₈₈N₅O₃₅[M+H⁺] 1450.5, found 1450.4.

EXAMPLE 12 Synthesis of Compound 20

The compound 8 (47 mg, 25 μmol) and 1.9 mg of bovine serum albumin weredissolved in HEPES buffer (50 mM, pH 5.0, 840 μL), and neuraminidase(manufactured by Sigma-Aldrich Corporation, from Viblio Cholerae, 369mU) was added thereto. This solution was allowed to stand at 37° C. for37 hours, and thereafter the termination of the reaction was confirmedby HPLC analysis. The reaction solution was lyophilized, and thelyophilized product was subsequently purified by HPLC (YMC Packed ColumnD-ODS-5 S-5 120A ODS No. 2020178, 20×250 mm, eluent: 50 mM aqueousammonium acetate:acetonitrile=80:20, flow rate: 4 mL/min). Further, theresidue was desalted on an ODS column (Cosmosil 75C₁₈-OPN, 15×100 mm,eluted first with 50 mL of H₂O and then with 25% acetonitrile), to givea desired compound 20 (26 mg, yield: 65%). The physical data for theresulting compound are as follows.

¹H-NMR (30° C.) δ7.92 (d, 2H, J=7.5 Hz, Fmoc), 7.71 (d, 2H, J=7.5 Hz,Fmoc), 7.51 (dd, 2H, J=7.5 Hz, Fmoc), 7.43 (dd, 2H, J=7.5 Hz, Fmoc),5.12 (s, 1H, Man4-H-1), 4.99 (d, 1H, J=9.4 Hz, GlcNAc1-H-1), 4.91 (s,1H, Man4′-H-1), 4.77 (s, 1H, Man3-H-1), 4.57 (bd, 2H, GlcNAc2,5′-H-1),4.46 (d, 1H, J=7.5 Hz, Gal6′-H-1), 4.34 (t, 3H, Fmoc), 4.24 (bs, 1H,Man4′-H-2), 4.19 (bs, 1H, Man4-H-2), 2.72 (bd, 1H, J=15.5 Hz, Asn-βCH),2.52 (bdd, 1H, J=9.2 Hz, 15.5 Hz, Asn-βCH), 2.06, 2.05, 1.89 (each s,each 3H, Ac); HRMS Calcd for C₆₇H₉₇N₅NaO₄₀[M+Na⁺] 1634.5608, found1634.5644.

EXAMPLE 13 Synthesis of Compound 71

The compound 20 (12 mg, 7.4 μmol) and 1.0 mg of bovine serum albuminwere dissolved in HEPES buffer (50 mM, pH 5.0, 330 μL), andβ-galactosidase (manufactured by Seikagaku Corporation, from Jack Beans,12 μL, 297 mU) was added thereto. This solution was allowed to stand at37° C. for 46 hours, and thereafter the termination of the reaction wasconfirmed by HPLC analysis. The reaction solution was purified by HPLC(YMC Packed Column D-ODS-5 S-5 120A ODS No. 2020178, 20×250 mm, eluent:50 mM aqueous ammonium acetate:acetonitrile=80:20, flow rate: 4 mL/min).Further, the residue was desalted on an ODS column (Cosmosil 75C₁₈-OPN,15×100 mm, eluted first with 50 mL of H₂O and then with 25%acetonitrile), to give a desired compound 71 (6.6 mg, yield: 61%). Thephysical data for the resulting compound are as follows.

¹H-NMR (30° C.) δ7.90 (d, 2H, J=7.5 Hz, Fmoc), 7.70 (d, 2H, J=7.5 Hz,Fmoc), 7.49 (dd, 2H, J=7.5 Hz, Fmoc), 7.42 (dd, 2H, J=7.5 Hz, Fmoc),5.11 (s, 1H, Man4-H-1), 4.99 (d, 1H, J=9.4 Hz, GlcNAc1-H-1), 4.91 (s,1H, Man4′-H-1), 4.76 (s, 1H, Man3-H-1), 4.55 (d, 2H, GlcNAc2,5-H-1),4.31 (b, 1H, Fmoc), 4.24 (bs, 1H, Man3-H-2), 4.18 (bs, 1H, Man4-H-2),3.97 (dd, 1H, J=1.8 Hz, 3.3 Hz, Man4′-H-2), 2.72 (bd, 1H, J=15.5 Hz,Asn-βCH), 2.52 (bdd, 1H, J=8.0 Hz, 15.5 Hz, Asn-βCH), 2.06, 2.05, 1.88(each s, each 3H, Ac); MS(Fab) Calcd for C₆₁H₈₈N₅O₃₅[M+H⁺] 1450.5, found1450.3.

EXAMPLE 14 Synthesis of Compound 16

The compound 5 (32 mg, 18.4 μmol) and 2.5 mg of bovine serum albuminwere dissolved in HEPES buffer (50 mM, pH 5.0, 713 μL), andneuraminidase (manufactured by Sigma-Aldrich Corporation, from ViblioCholerae, 134 mU) was added thereto. This solution was allowed to standat 37° C. for 17 hours, and thereafter the termination of the reactionwas confirmed by HPLC analysis. Subsequently, the reaction solution waspurified by HPLC (YMC Packed Column D-ODS-5 S-5 120A ODS No. 2020178,20×250 mm, eluent: 50 mM aqueous ammonium acetate:acetonitrile=80:20,flow rate: 4 mL/min). Further, the residue was desalted on an ODS column(Cosmosil 75C₁₈-OPN, 15×100 mm, eluted first with 50 mL of H₂O and thenwith 25% acetonitrile), to give a desired compound 16 (13 mg, yield:52%). The physical data for the resulting compound are as follows.

¹H-NMR (30° C.) δ7.92 (d, 2H, J=7.5 Hz, Fmoc), 7.71 (d, 2H, J=7.5 Hz,Fmoc), 7.51 (dd, 2H, J=7.5 Hz, Fmoc), 7.44 (dd, 2H, J=7.5 Hz, Fmoc),5.00 (d, 1H, J=9.9 Hz, GlcNAc1-H-1), 4.92 (s, 1H, Man4′-H-1), 4.75 (s,1H, Man3-H-1), 4.58 (d, 2H, J=7.5 Hz, GlcNAc2,5′-H-1), 4.47 (d, 1H,J=7.8 Hz, Gal6′-H-1), 4.34 (t, 1H, Fmoc), 4.10 (bd, 1H, Man3-H-2), 4.07(bs, 1H, Man4′-H-2), 2.72 (bdd, 1H, J=15.5 Hz, Asn-βCH), 2.52 (bdd, 1H,J=9.2 Hz, 15.5 Hz, Asn-βCH), 2.07, 2.05, 1.89 (each s, each 3H, Ac);MS(Fab) Calcd for C₆₁H₈₈N₅O₃₅[M+H⁺] 1450.5, found 1450.3.

EXAMPLE 15 Synthesis of Compound 17

The compound 16 (9 mg, 6.2 μmol) and 1.6 mg of bovine serum albumin weredissolved in HEPES buffer (50 mM, pH 5.0, 613 μL), and β-galactosidase(manufactured by Seikagaku Corporation, from Jack Beans, 186 mU) wasadded thereto. This solution was allowed to stand at 37° C. for 32hours, and thereafter the termination of the reaction was confirmed byHPLC analysis. The reaction solution was purified by HPLC (YMC PackedColumn D-ODS-5 S-5 120A ODS No. 2020178, 20×250 mm, eluent: 50 mMaqueous ammonium acetate:acetonitrile=80:20, flow rate: 4 mL/min).Further, the residue was desalted on an ODS column (Cosmosil 75C₁₈-OPN,15×100 mm, eluted first with 50 mL of H₂O and then with 25%acetonitrile), to give a desired compound 17 (5.4 mg, yield: 68%). Thephysical data for the resulting compound are as follows.

¹H-NMR (30° C.) δ7.89 (d, 2H, J=7.5 Hz, Fmoc), 7.68 (d, 2H, J=7.5 Hz,Fmoc), 7.49 (dd, 2H, J=7.5 Hz, Fmoc), 7.42 (dd, 2H, J=7.5 Hz, Fmoc),4.99 (d, 1H, J=9.7 Hz, GlcNAc1-H-1), 4.91 (s, 1H, Man4′-H-1), 4.55 (d,1H, J=8.1 Hz, GlcNAc2,5′-H-1), 4.09, 4.07 (s, 1H, Man4′-H-2, Man3-H-2),2.72 (bd, 1H, J=15.5 Hz Asn-βCH), 2.56 (bdd, 1H, J=8.1 Hz, 15.5 Hz,Asn-βCH), 2.07, 2.05, 1.89 (each s, each 3H, Ac); MS(Fab) Calcd forC₅₅H₇₇N₅NaO₃₀[M+Na⁺] 1310.5, found 1310.2.

EXAMPLE 16 Synthesis of Compound 18

The compound 17 (3.4 mg, 2.6 μmol) and 1.1 mg of bovine serum albuminwere dissolved in HEPES buffer (50 mM, pH 5.0, 257 μL), andN-acetyl-β-D-glucosaminidase (Sigma-Aldrich Corporation, from JackBeans, 144 mU) was added thereto. This solution was allowed to stand at37° C. for 24 hours, and thereafter the termination of the reaction wasconfirmed by HPLC analysis. The reaction solution was purified by HPLC(YMC Packed Column D-ODS-5 S-5 120A ODS No. 2020178, 20×250 mm, eluent:50 mM aqueous ammonium acetate:acetonitrile=80:20, flow rate: 4 mL/min).Further, the residue was desalted on an ODS column (Cosmosil 75C₁₈-OPN,15×100 mm, eluted first with 50 mL of H₂O and then with 25%acetonitrile), to give a desired compound 18 (2.1 mg, yield: 75%). Thephysical data for the resulting compound are as follows.

¹H-NMR (30° C.) δ7.91 (d, 2H, J=7.5 Hz, Fmoc), 7.71 (d, 2H, J=7.5 Hz,Fmoc), 7.51 (dd, 2H, J=7.5 Hz, 7.5 Hz, Fmoc), 7.43 (dd, 2H, J=7.5 Hz,7.5 Hz, Fmoc), 5.00 (d, 1H, J=9.7 Hz, GlcNAc1-H-1), 4.91 (d, 1H, J=1.6Hz, Man4′-H-1), 4.76 (s, 1H, Man3-H-1), 4.58 (d, 1H, J=7.8 Hz,GlcNAc2-H-1), 4.34 (t, 1H, Fmoc), 4.07 (d, 1H, J=2.7 Hz, Man4′-H-2),3.97 (dd, 1H, J=1.6 Hz, 3.7 Hz, Man3-H-2), 2.72 (bdd, 1H, J=3.2 Hz, 15.1Hz, Asn-βCH), 2.52 (bdd, 1H, J=8.9 Hz, 15.1 Hz, Asn-βCH), 2.07, 1.89(each s, each 3H, Ac); MS(Fab) Calcd for C₄₇H₆₅N₄O₂₅[M+Na⁺] 1085.4,found 1085.3.

EXAMPLE 17 Synthesis of Compound 21

The compound 9 (28 mg, 16 μmol) and 1.7 mg of bovine serum albumin weredissolved in HEPES buffer (50 mM, pH 5.0, 624 μL), and neuraminidase(manufactured by Sigma-Aldrich Corporation, from Viblio Cholerae, 117mU) was added thereto. This solution was allowed to stand at 37° C. for17 hours, and thereafter the termination of the reaction was confirmedby HPLC analysis. Subsequently, the reaction solution was purified byHPLC (YMC Packed Column D-ODS-5 S-5 120A ODS No. 2020178, 20×250 mm,eluent: 50 mM aqueous ammonium acetate:acetonitrile=80:20, flow rate: 4mL/min). Further, the residue was desalted on an ODS column (Cosmosil75C₁₈-OPN, 15×100 mm, eluted first with 50 mL of H₂O and then with 25%acetonitrile), to give a desired compound 21 (14.6 mg, yield: 68%). Thephysical data for the resulting compound are as follows.

¹H-NMR (30° C.) δ7.92 (d, 2H, J=7.5 Hz, Fmoc), 7.71 (d, 2H, J=7.5 Hz,Fmoc), 7.50 (dd, 2H, J=7.5 Hz, Fmoc), 7.43 (dd, 2H, J=7.5 Hz, Fmoc),5.12 (s, 1H, Man4-H-1), 4.99 (d, 1H, J=9.5 Hz, GlcNAc1-H-1), 4.77 (s,1H, Man3-H-1), 4.57 (d, 2H, J=7.2 Hz, GlcNAc2-H-1), 4.46 (d, 1H, J=7.8Hz, Gal6-H-1), 4.34 (t, 1H Fmoc), 4.22 (bd, 1H, J=2.7 Hz, Man3-H-2),4.19 (b, 1H, Man4-H-2), 2.72 (bdd, 1H, J=15.5 Hz, Asn-βCH), 2.52 (bdd,1H, J=9.8 Hz, 15.5 Hz, Asn-βCH), 2.05 (s, 6H, Ac×2), 1.89 (s, 3H, Ac);MS(Fab) Calcd for C₆₁H₈₈N₅O₃₅[M+H⁺] 1450.5, found 1450.3.

EXAMPLE 18 Synthesis of Compound 22

The compound 21 (10 mg, 6.9 μmol) and 1.6 mg of bovine serum albuminwere dissolved in HEPES buffer (50 mM, pH 5.0, 672 μL), andβ-galactosidase (manufactured by Seikagaku Corporation, from Jack Beans,205 mU) was added thereto. This solution was allowed to stand at 37° C.for 20 hours, and thereafter the termination of the reaction wasconfirmed by HPLC analysis. The reaction solution was purified by HPLC(YMC Packed Column D-ODS-5 S-5 120A ODS No. 2020178, 20×250 mm, eluent:50 mM aqueous ammonium acetate:acetonitrile=80:20, flow rate: 4 mL/min).Further, the residue was desalted on an ODS column (Cosmosil 75C₁₈-OPN,15×100 mm, eluted first with 50 mL of H₂O and then with 25%acetonitrile), to give a desired compound 22 (5.6 mg, yield: 64%). Thephysical data for the resulting compound are as follows.

¹H-NMR (30° C.) δ7.87 (d, 2H, J=7.5 Hz, Fmoc), 7.67 (d, 2H, J=7.5 Hz,Fmoc), 7.48 (dd, 2H, J=7.5 Hz, Fmoc), 7.41 (dd, 2H, J=7.5 Hz, Fmoc),5.12 (s, 1H, Man4-H-1), 4.99 (d, 1H, J=9.7 Hz, GlcNAc1-H-1), 4.76 (s,1H, Man3-H-1), 4.55 (d, 2H, J=8.6 Hz, GlcNAc2,5-H-1), 4.26 (t, 1H,Fmoc), 4.22 (d, 1H, J=2.2 Hz, Man3-H-2), 4.18 (bdd, 1H, J=1.3 Hz, 3.3Hz, Man4-H-2), 2.72 (bd, 1H, J=15.5 Hz, Asn-βCH), 2.54 (bdd, 1H, J=9.5Hz, 15.5 Hz, Asn-βCH), 2.05 (s, 6H, Ac×2), 1.88 (s, 3H, Ac); MS(Fab)Calcd for C₅₅H₇₈N₅O₃₀[M+H⁺] 1288.5, found 1288.3.

EXAMPLE 19 Synthesis of Compound 23

The compound 22 (3.6 mg, 2.8 μmol) and 1.2 mg of bovine serum albuminwere dissolved in HEPES buffer (50 mM, pH 5.0, 277 μL), andN-acetyl-β-D-glucosaminidase (Sigma-Aldrich Corporation, from JackBeans, 195 mU) was added thereto. This solution was allowed to stand at37° C. for 24 hours, and thereafter the termination of the reaction wasconfirmed by HPLC analysis. The reaction solution was purified by HPLC(YMC Packed Column D-ODS-5 S-5 120A ODS No. 2020178, 20×250 mm, eluent:50 mM aqueous ammonium acetate:acetonitrile=80:20, flow rate: 4 mL/min).Further, the residue was desalted on an ODS column (Cosmosil 75C₁₈-OPN,15×100 mm, eluted first with 50 mL of H₂O and then with 25%acetonitrile), to give a desired compound 23 (2.3 mg, yield: 77%). Thephysical data for the resulting compound are as follows.

¹H-NMR (30° C.) δ7.91 (d, 2H, J=7.5 Hz, Fmoc), 7.70 (d, 2H, J=7.5 Hz,Fmoc), 7.50 (dd, 2H, J=7.5 Hz, Fmoc), 7.43 (dd, 2H, J=7.5 Hz, Fmoc),5.11 (s, 1H, Man4-H-1), 4.99 (d, 1H,J=9.7 Hz, GlcNAc1-H-1), 4.77 (s, 1H,Man3-H-1), 4.57 (d, 1H, J=6.5 Hz, GlcNAc-H-1), 4.33 (t, 1H, Fmoc), 4.22(d, 1H, J=3.0 Hz, Man3-H-2), 4.07 (bdd, 1H, J=2.1 Hz, Man4-H-2), 2.72(bdd, 1H, J=15.5 Hz, Asn-βCH), 2.52 (bdd, 1H, J=8.9 Hz, 15.5 Hz,Asn-βCH), 2.05, 1.89 (each s, each 3H, Ac); MS(Fab) Calcd forC₄₇H₆₅N₄O₂₅[M+H⁺] 1085.4, found 1085.3.

EXAMPLE 20 Synthesis of Compound 11

The compound 10 (123 mg, 62 μmol) and bovine serum albumin (1.1 mg) weredissolved in HEPES buffer (50 mM, pH 5.0, 2.5 mL), and β-galactosidase(manufactured by Seikagaku Corporation, from Jack Beans, 24 μL, 612 mU)was added thereto. This solution was allowed to stand at 37° C. for 61hours, and thereafter the termination of the reaction was confirmed byHPLC analysis. The reaction solution was lyophilized, and subsequentlypurified by HPLC (YMC Packed Column D-ODS-5 S-5 120A ODS No. 2020178,20×250 mm, eluent: 50 mM aqueous ammonium acetate:acetonitrile=80:20,flow rate: 3.5 mL/min). Further, the residue was desalted on an ODScolumn (Cosmosil 75C₁₈-OPN, 15×100 mm, eluted first with 50 mL of H₂Oand then with 25% acetonitrile), to give a desired compound 11 (71 mg,yield: 70%). The physical data for the resulting compound are asfollows.

¹H-NMR (30° C.) δ7.91 (d, 2H, J=7.5 Hz, Fmoc), 7.71 (d, 2H, J=7.5 Hz,Fmoc), 7.50 (dd, 2H, J=7.5 Hz, Fmoc), 7.43 (dd, 2H, J=7.5 Hz, Fmoc),5.11 (s, 1H, Man4-H-1), 4.99 (1H, d, J=9.9 Hz, GlcNAc1-H-1), 4.91 (s,1H, Man4′-H-1), 4.76 (s, 1H, Man3-H-1), 4.55 (d, 2H, J=8.6 Hz,GlcNAc2,5-H-1), 4.34 (t, 1H, Fmoc), 4.24 (s, 1H, Man3-H-2), 4.18 (s, 1H,Man4-H-2), 4.10 (s, 1H, Man4′-H-2) 2.72 (bd, 1H, J=15.5 Hz, Asn-βCH),2.51 (bdd, 1H, J=9.0 Hz, 15.5 Hz, Asn-βCH), 2.06 (s, 3H, Ac), 2.05 (s,6H, Ac×2), 1.88 (s, 3H, Ac); HRMS Calcd for C₆₉H₁₀₀N₆NaO₄₀[M+Na⁺]1675.5873, found 1675.5841.

EXAMPLE 21 Synthesis of Compound 12

The compound 11 (50 mg, 30 μmol) and 2.0 mg of bovine serum albumin weredissolved in HEPES buffer (50 mM, pH 5.0, 920 μL), andN-acetyl-β-D-glucosaminidase (manufactured by Sigma-Aldrich Corporation,from Jack Beans, 2.1 U) was added thereto. This solution was allowed tostand at 37° C. for 48 hours, and thereafter the termination of thereaction was confirmed by HPLC analysis. The reaction solution waspurified by HPLC (YMC Packed Column D-ODS-5 S-5 120A ODS No. 2020178,20×250 mm, eluent: 50 mM aqueous ammonium acetate:acetonitrile=80:20,flow rate: 4 mL/min), and lyophilized. This residue was desalted on anODS column (Cosmosil 75C₁₈-OPN, 15×100 mm, eluted first with 50 mL ofH₂O and then with 25% acetonitrile), to give a desired compound 12 (25mg, yield: 66%). The physical data for the resulting compound are asfollows.

¹H-NMR (30° C.) δ7.91 (d, 2H, J=7.5 Hz, Fmoc), 7.70 (d, 2H, J=7.5 Hz,Fmoc), 7.50 (dd, 2H, J=7.5 Hz, Fmoc), 7.43 (dd, 2H, J=7.5 Hz, Fmoc),5.10 (s, 1H, Man4-H-1), 4.99 (d, 1H, J=9.7 Hz, GlcNAc1-H-1), 4.91 (bd,1H, J=1.6 Hz, Man4′-H-1), 4.77 (s, 1H, Man3-H-1), 4.58–4.52 (b, 1H,GlcNAc2-H-1), 4.33 (t, 1H, Fmoc), 4.24 (bs, 1H, Man3-H-2), 4.06 (dd, 1H,J=1.6 Hz, 3.2 Hz, Man4-H-2), 3.97 (dd, 1H, J=1.6 Hz, 3.5 Hz, Man4′-H-2),2.72 (bd, 1H, J=15.5 Hz, Asn-βCH), 2.53 (bdd, 1H, J=9.0 Hz, 15.5 Hz,Asn-βCH), 2.05, 1.88 (each s, each 3H, Ac).

EXAMPLE 22 Synthesis of Compound 13

The compound 12 (10 mg, 11 μmol) and 0.9 mg of bovine serum albumin weredissolved in HEPES buffer (50 mM, pH 5.0, 440 μL), and α-mannosidase(manufactured by Sigma-Aldrich Corporation, from Jack Beans, 30 μL, 3.2U) was added thereto. This solution was allowed to stand at 37° C. for21 hours, and thereafter the termination of the reaction was confirmedby HPLC analysis. Subsequently, the reaction solution was purified byHPLC (YMC Packed Column D-ODS-5 S-5 120A ODS No. 2020178, 20×250 mm,eluent: 50 mM aqueous ammonium acetate:acetonitrile=80:20, flow rate: 4mL/min). Further, the residue was desalted on an ODS column (Cosmosil75C₁₈-OPN, 15×100 mm, eluted first with 50 ml of H₂O and then with 25%acetonitrile), to give a desired compound 13 (3 mg, yield: 43%). Thephysical data for the resulting compound are as follows.

¹H-NMR (30° C.) δ7.92 (d, 2H, J=7.5 Hz, Fmoc), 7.71 (d, 2H, J=7.5 Hz,Fmoc), 7.51 (dd, 2H, J=7.5 Hz, Fmoc), 7.43 (dd, 2H, J=7.5 Hz, Fmoc),4.99 (d, 1H, J=9.5 Hz, GlcNAc1-H-1), 4.76 (s, 1H, Man3-H-1), 4.57 (1H,GlcNAc2-H-1), 4.06 (d, 1H, J=3.2 Hz, Man3-H-2), 2.72 (bd, 1H, J=15.5 Hz,Asn-βCH), 2.52 (bdd, 1H, J=8.3 Hz, 15.5 Hz, Asn-βCH), 2.05, 1.89 (eachs, each 3H, Ac).

(Deprotection of Fmoc Group of Sugar Chain Asparagine Derivative)

All of the sugar chain asparagine derivatives were subjected to thedeprotection of the Fmoc group in accordance with the followingprocedures. First, 240 μL of N,N-dimethylformamide and 160 μL ofmorpholine were added per 1 μmol of the Fmoc form of the sugar chainasparagine, and the resulting mixture was subjected to reaction at roomtemperature under argon atmosphere. The termination of the reaction wasconfirmed by TLC (eluent: 1M ammonium acetate:isopropanol=8:5), andthereafter the mixture was cooled with ice water. To this mixture wasadded diethyl ether in an amount of 10 times that of the reactionsolution, with stirring the mixture for 15 minutes, and thereafter theprecipitates formed were filtered. The residue obtained was dissolved inwater, and evaporated at 35° C. Further, a procedure of adding 3 mL oftoluene thereto and evaporating the mixture was repeated three times.The residue was purified by reverse phase column chromatography(Cosmosil 75C₁₈-OPN, 15×100 mm, eluent: water).

EXAMPLE 23 Synthesis of Compound 33

The compound 10 (10.5 mg, 5.3 μmol) was reacted for 7 hours inaccordance with the above procedures, to give a desired compound 33 (7mg, yield: 76%). The resulting compound was confirmed from the findingthat its ¹H-NMR was identical to that of the preparation.

EXAMPLE 24 Synthesis of Compound 26

The compound 3 (8.0 mg, 3.8 μmol) was subjected to the reaction for 21hours in accordance with the above procedures, to give a desiredcompound 26 (6.3 mg, yield: 88%). The physical data for the resultingcompound are as follows.

¹H-NMR (30° C.) δ5.13 (s, 1H, Man4-H-1), 5.07 (d, 1H, J=9.9 Hz,GlcNAc1-H-1), 4.95 (s, 1H, Man4′-H-1), 4.78 (s, 1H, Man3-H-1), 4.62 (2H,GlcNAc2,5′-H-1), 4.56 (d, 1H, J=8.1 Hz, GlcNAc5-H-1), 4.52 (d, 1H, J=7.8Hz, Gal6′-H-1), 4.25 (bs, 1H, Man3-H-2), 4.19 (bs, 1H, Man4′-H-2), 4.12(bs, 1H, Man4-H-2), 2.94 (dd, 1H, J=4.5 Hz, 17.0 Hz, Asn-βCH), 2.85 (dd,1H, J=6.8 Hz, 17.0 Hz, Asn-βCH), 2.68 (dd, 1H, J=4.6 Hz, 12.4 Hz,NeuAc7′-H-3_(eq)), 2.08, 2.07, 2.06, 2.04, 2.02 (each s, each 3H, Ac),1.72 (dd, 1H, J=12.1 Hz, 12.1 Hz, NeuAc7′-H-3_(ax)); MS(Fab) Calcd forC₇₁H₁₁₈N₇O₅₁[M+H⁺] 1884.7, found 1884.5.

EXAMPLE 25 Synthesis of Compound 27

The compound 4 (11.0 mg, 5.8 μmol) was subjected to the reaction for 23hours in accordance with the above procedures, to give a desiredcompound 27 (8.5 mg, yield: 88%). The physical data for the resultingcompound are as follows.

¹H-NMR (30° C.) δ5.11 (s, 1H, Man4-H-1), 5.08 (d, 1H, J=9.7 Hz,GlcNAc1-H-1), 4.95 (s, 1H, Man4′-H-1), 4.78 (s, 1H, Man3-H-1), 4.62 (d,2H, GlcNAc2,5′-H-1), 4.45 (d, 1H, J=7.6 Hz, Gal6′-H-1), 4.26 (bd, 1H,Man3-H-2), 4.12 (bd, 1H, Man4′-H-2), 4.08 (bdd, 1H, J=1.6 Hz, 3.3 Hz,Man4-H-2), 2.94 (dd, 1H, J=4.0 Hz, 17.2 Hz, Asn-βCH), 2.85 (dd, 1H,J=7.2 Hz, 17.2 Hz, Asn-βCH), 2.68 (dd, 1H, J=4.1 Hz, 12.1 Hz,NeuAc7′-H-3_(eq)), 2.09, 2.07, 2.04, 2.02 (each s, each 3H, Ac), 1.72(dd, 1H, J=12.1 Hz, 12.1 Hz, NeuAc7′-H-3_(ax)); MS(Fab) Calcd forC₆₃H₁₀₄N₆NaO₄₆[M+Na⁺] 1703.6, found 1703.1.

EXAMPLE 26 Synthesis of Compound 28

The compound 5 (7.0 mg, 4.0 μmol) was subjected to the reaction for 21hours in accordance with the above procedures, to give a desiredcompound 28 (5.3 mg, yield: 87%). The physical data for the resultingcompound are as follows.

¹H-NMR (30° C.) δ5.07 (d, 1H, J=9.4 Hz, GlcNAc1-H-1), 4.94 (s, 1H,Man4′-H-1), 4.76 (s, 1H, Man3-H-1), 4.61, 4.59 (each d, each 1H,GlcNAc2,5′-H-1), 4.44 (d, 1H, J=7.8 Hz, Gal6′-H-1), 4.10, 4.07 (each 1H,Man4′,3-H-2), 2.93 (dd, 1H, J=4.6 Hz, 17.5 Hz, Asn-βCH), 2.85 (dd, 1H,J=7.0 Hz, 17.5 Hz, Asn-βCH), 2.67 (dd, 1H, J=4.6 Hz, 12.2 Hz,NeuAc7′-H-3_(eq)), 2.08, 2.06, 2.02, 2.01 (each s, each 3H, Ac), 1.71(2H, dd, J=12.2 Hz, 12.2 Hz, NeuAc7′-H-3_(ax)); MS(Fab) Calcd forC₅₇H₉₄N₆NaO₄₁[M+Na⁺] 1541.5, found 1541.3.

EXAMPLE 27 Synthesis of Compound 30

The compound 7 (13.9 mg, 6.6 μmol) was subjected to the reaction for 7hours in accordance with the above procedures, to give a desiredcompound 30 (8.0 mg, yield: 64%). The physical data for the resultingcompound are as follows.

¹H-NMR (30° C.) δ5.13 (s, 1H, Man4-H-1), 5.06 (d, 1H, J=9.9 Hz,GlcNAc1-H-1), 4.91 (s, 1H, Man4′-H-1), 4.77 (s, 1H, Man3-H-1), 4.61,4.60 (each d, each 1H, J=8.0 Hz, GlcNAc2,5-H-1), 4.55 (d, 1H, J=8.4 Hz,GlcNAc5′-H-1), 4.44 (d, 1H, J=8.0 Hz, Gal6-H-1), 4.24 (bd, 1H,Man3-H-2), 4.19 (bdd, 1H, J=1.3 Hz, 3.2 Hz, Man4′-H-2), 4.10 (bdd, 1H,J=1.4 Hz, 3.2 Hz, Man4-H-2), 2.90 (dd, 1H, J=4.5 Hz, 16.7 Hz, Asn-βCH),2.80 (dd, 1H, J=7.5 Hz, 16.7 Hz, Asn-βCH), 2.66 (dd, 1H, J=4.6 Hz, 12.4Hz, NeuAc7-H-3_(eq)), 2.07, 2.06, 2.05, 2.02, 2.01 (each s, each 3H,Ac), 1.71 (dd, 1H, J=12.4 Hz, 12.4 Hz, NeuAc7-H-3_(ax)); MS(Fab) Calcdfor C₇₁H₁₁₇N₇NaO₅₁[M+Na⁺] 1906.7, found 1906.1.

EXAMPLE 28 Synthesis of Compound 31

The compound 8 (8.0 mg, 4.2 μmol) was subjected to the reaction for 12hours in accordance with the above procedures, to give a desiredcompound 31 (6.0 mg, yield: 86%). The physical data for the resultingcompound are as follows.

¹H-NMR (30° C.) δ5.12 (s, 1H, Man4-H-1), 5.06 (d, 1H, J=9.5 Hz,GlcNAc1-H-1), 4.91 (s, 1H, Man4′-H-1), 4.77 (s, 1H, Man3-H-1), 4.61,4.59 (each d, each 1H, GlcNAc2,5-H-1), 4.43 (d, 1H, J=8.0 Hz, Gal6-H-1),4.24 (bd, 1H, Man3-H-2), 4.18 (bdd, 1H, Man4′-H-2), 2.91 (bd, 1H, J=17.0Hz, Asn-βCH), 2.81 (dd, 1H, J=6.5 Hz, 17.0 Hz, Asn-βCH), 2.66 (dd, 1H,J=4.6 Hz, 12.6 Hz, NeuAc7-H-3_(eq)), 2.06, 2.06, 2.02, 2.00 (each s,each 3H, Ac), 1.70 (dd, 1H, J=12.6 Hz, 12.6 Hz, NeuAc7-H-3_(ax));MS(Fab) Calcd for C₆₃H₁₀₄N₆NaO₄₆[M+Na⁺] 1703.6, found 1703.0.

EXAMPLE 29 Synthesis of Compound 32

The compound 9 (7.7 mg, 4.4 μmol) was subjected to the reaction for 23hours in accordance with the above procedures, to give a desiredcompound 32 (5.2 mg, yield: 78%). The physical data for the resultingcompound are as follows.

¹H-NMR (30° C.) δ5.14 (s, 1H, Man4-H-1), 5.07 (d, 1H, J=9.4 Hz,GlcNAc1-H-1), 4.78 (s, 1H, Man3-H-1), 4.61, 4,60 (each d, each 1H,GlcNAc2,5-H-1), 4.44 (d, 1H, J=8.0 Hz, Gal6-H-1), 4.23 (d, 1H, J=3.0 Hz,Man3-H-2), 4.19 (bdd, 1H, J=1.3 Hz, 2.9 Hz, Man4-H-2), 2.92 (dd, 1H,J=4.1 Hz, 17.2 Hz, Asn-βCH), 2.83 (dd, 1H, J=7.5 Hz, 12.7 Hz, Asn-βCH),2.67 (dd, 1H, J=4.6 Hz, 12.7 Hz, NeuAc7-H-3_(eq)), 2.06 (s, 6H, Ac×2),2.03, 2.01 (each s, each 3H, Ac), 1.71 (dd, 1H, J=12.7 Hz, 12.7 Hz,NeuAc7-H-3_(ax)); MS(Fab) Calcd for C₅₇H₉₄N₆NaO₄₁[M+Na⁺] 1541.5, found1541.2.

EXAMPLE 30 Synthesis of Compound 37

The compound 14 (9.1 mg, 5.0 μmol) was subjected to the reaction for 13hours in accordance with the above procedures, to give a desiredcompound 37 (6.5 mg, yield: 77%). The physical data for the resultingcompound are as follows.

¹H-NMR (30° C.) δ5.11 (s, 1H, Man4-H-1), 5.06 (d, 1H, J=9.5 Hz,GlcNAc1-H-1), 4.92 (s, 1H, Man4′-H-1), 4.75 (s, 1H, Man3-H-1), 4.61,4.57, 4.55 (each d, each 1H, J=7.5 Hz, GlcNAc2,5,5′-H-1), 4.46 (d, 1H,J=7.3 Hz, Gal6′-H-1), 4.23 (bs, 1H, Man3-H-2), 4.18 (bs, 1H, Man4′-H-2),4.10 (bs, 1H, Man4-H-2), 2.87 (dd, 1H, J=4.8 Hz, 17.0 Hz, Asn-βCH), 2.76(dd, 1H, J=7.2 Hz, 17.0 Hz, Asn-βCH), 2.07 (s, 3H, Ac), 2.04 (s, 6H,Ac×2), 2.00 (s, 3H, Ac); MS(Fab) Calcd for C₆₀H₁₀₀N₆NaO₄₃[M+Na⁺] 1615.6,found 1615.0.

EXAMPLE 31 Synthesis of Compound 42

The compound 19 (9.8 mg, 5.4 μmol) was subjected to the reaction for 13hours in accordance with the above procedures, to give a desiredcompound 42 (8.0 mg, yield: 88%). The physical data for the resultingcompound are as follows.

¹H-NMR (30° C.) δ5.11 (s, 1H, Man4-H-1), 5.06 (d, 1H,J=9.5 Hz,GlcNAc1-H-1), 4.91 (s, 1H, Man4′-H-1), 4.76 (s, 1H, Man3-H-1), 4.60,4.57, 4.55 (each d, each 1H, GlcNAc2,5,5′-H-1), 4.46 (d, 1H, J=7.8 Hz,Gal6-H-1), 4.28 (s, 1H, Man3-H-2), 4.18 (s, 1H, Man4′-H-2), 4.10 (s, 1H,Man4-H-2), 2.88 (dd, 1H, J=4.0 Hz, 16.6 Hz, Asn-βCH), 2.77 (dd, 1H,J=7.5 Hz, 16.6 Hz, Asn-βCH), 2.07 (s, 3H, Ac), 2.04 (s, 6H, Ac×2), 2.00(s, 3H, Ac); MS(Fab) Calcd for C₆₀H₁₀₁N₆O₄₃[M+H⁺] 1593.6, found 1593.8.

EXAMPLE 32 Synthesis of Compound 38

The compound 15 (5.1 mg, 3.2 μmol) was subjected to the reaction for 11hours in accordance with the above procedures, to give a desiredcompound 38 (4.0 mg, yield: 91%). The physical data for the resultingcompound are as follows.

¹H-NMR (30° C.) δ5.10 (s, 1H, Man4-H-1), 5.07 (d, 1H, J=9.4 Hz,GlcNAc1-H-1), 4.92 (s, 1H, Man4′-H-1), 4.76 (s, 1H, Man3-H-1), 4.61,4.57 (each d, each 1H, J=7.8 Hz, GlcNAc2,5′-H-1), 4.47 (d, 1H, J=7.8 Hz,Gal6′-H-1), 4.24 (d, 1H, J=2.3 Hz, Man3-H-2), 4.10, 4.06 (each bd, each1H, Man4′,4-H-2), 2.90 (dd, 1H, J=4.2 Hz, 16.8 Hz, Asn-βCH), 2.81 (dd,1H, J=7.3 Hz, 16.8 Hz, Asn-βCH), 2.07, 2.04, 2.01 (each s, each 3H, Ac);MS(Fab) Calcd for C₅₂H₈₈N₅O₃₈[M+H⁺] 1390.5, found 1390.1.

EXAMPLE 33 Synthesis of Compound 72

The compound 70 (4.0 mg, 2.8 μmol) was subjected to the reaction for 13hours in accordance with the above procedures, to give a desiredcompound 72 (2.9 mg, yield: 85%). The physical data for the resultingcompound are as follows.

¹H-NMR (30° C.) δ5.09 (s, 1H, Man4-H-1), 5.06 (d, 1H, J=9.8 Hz,GlcNAc1-H-1), 4.91 (s, 1H, Man4′-H-1), 4.76 (s, 1H, Man3-H-1), 4.61,4.54 (each d, each 1H, GlcNAc2,5-H-1), 4.24 (s, 1H, Man3-H-2), 4.10,4.06 (each bs, each 1H, Man4,4′-H-2), 2.87 (dd, 1H, J=17.2 Hz, Asn-βCH),2.76 (dd, 1H, J=6.5 Hz, 17.2 Hz, Asn-βCH), 2.07, 2.04, 2.00 (each s,each 3H, Ac); MS(Fab) Calcd for C₄₆H₇₈N₅O₃₃[M+H⁺] 1228.5, found 1228.3.

EXAMPLE 34 Synthesis of Compound 43

The compound 20 (5.4 mg, 3.3 μmol) was subjected to the reaction for 11hours in accordance with the above procedures, to give a desiredcompound 43 (4.1 mg, yield: 87%). The physical data for the resultingcompound are as follows.

¹H-NMR (30° C.) δ5.11 (s, 1H, Man4-H-1), 5.07 (d, 1H, J=9.5 Hz,GlcNAc1-H-1), 4.91 (s, 1H, Man4′-H-1), 4.77 (s, 1H, Man3-H-1), 4.61,4.57 (each d, each 1H, GlcNAc2,5-H-1), 4.46 (d, 1H, Gal6-H-1), 4.24 (s,1H, Man3-H-2), 4.18 (bs, 1H, Man4-H-2), 2.90 (dd, 1H, J=4.0 Hz, 17.0 Hz,Asn-βCH), 2.80 (dd, 1H, J=7.3 Hz, 17.0 Hz, Asn-βCH), 2.07, 2.04, 2.01(each s, each 3H, Ac); MS(Fab) Calcd for C₅₂H₈₈N₅O₃₈[M+H⁺] 1390.5, found1390.2.

EXAMPLE 35 Synthesis of Compound 73

The compound 71 (4.0 mg, 2.8 μmol) was subjected to the reaction for 13hours in accordance with the above procedures, to give a desiredcompound 73 (2.9 mg, yield: 85%). The physical data for the resultingcompound are as follows.

¹H-NMR (30° C.) δ5.11 (s, 1H, Man4-H-1), 5.06 (d, 1H,J=9.9 Hz,GlcNAc1-H-1), 4.91 (s, 1H, Man4′-H-1), 4.77 (s, 1H, Man3-H-1), 4.60,4.54 (each d, each 1H, J=7.9 Hz, GlcNAc2,5-H-1), 4.24 (s, 1H, Man3-H-2),4.18 (dd, 1H, J=1.6 Hz, 1.6 Hz, Man4-H-2), 3.96 (1H, dd, J=1.6 Hz, 1.6Hz, Man4-H-2), 2.88 (dd, 1H, J=4.3 Hz, 16.8 Hz, Asn-βCH), 2.77 (dd, 1H,J=7.2 Hz, 16.8 Hz, Asn-βCH), 2.06, 2.04, 2.00 (each s, each 3H, Ac);MS(Fab) Calcd for C₄₆H₇₈N₅O₃₃[M+H⁺] 1228.5, found 1228.3.

EXAMPLE 36 Synthesis of Compound 39

The compound 16 (2.2 mg, 1.5 μmol) was subjected to the reaction for 7hours in accordance with the above procedures, to give a desiredcompound 39 (1.6 mg, yield: 84%). The physical data for the resultingcompound are as follows.

¹H-NMR (30° C.) δ5.07 (d, 1H, J=9.7 Hz, GlcNAc1-H-1), 4.92 (s, 1H,Man4′-H-1), 4.75 (s, 1H, Man3-H-1), 4.62, 4.58 (each d, each 1H,GlcNAc2,5-H-1), 4.09, 4.08 (each s, each 1H, Man3,4′-H-2), 2.91 (dd, 1H,J=4.1 Hz, 16.9 Hz, Asn-βCH), 2.81 (dd, 1H, J=6.8 Hz, 16.9 Hz, Asn-βCH),2.08, 2.04, 2.01 (each s, each 3H, Ac); MS(Fab) Calcd forC₄₆H₇₇N₅NaO₃₃[M+Na⁺] 1250.4, found 1250.3.

EXAMPLE 37 Synthesis of Compound 40

The compound 17 (1.5 mg, 1.2 μmol) was subjected to the reaction for 14hours in accordance with the above procedures, to give a desiredcompound 40 (1.1 mg, yield: 89%). The physical data for the resultingcompound are as follows.

¹H-NMR (30° C.) δ5.07 (d, 1H, J=9.5 Hz, GlcNAc1-H-1), 4.91 (s, 1H,Man4′-H-1), 4.76 (s, 1H, Man3-H-1), 4.62, 4.55 (each d, each 1H,GlcNAc2,5-H-1), 4.10, 4.07 (each s, each 1H, Man4′,3-H-2), 2.89 (dd, 1H,J=3.7 Hz, 17.0 Hz, Asn-βCH), 2.79 (dd, 1H, J=7.0 Hz, 17.0 Hz, Asn-βCH),2.07, 2.05, 2.01 (each s, each 3H, Ac); MS(Fab) Calcd forC₄₀H₆₇N₅NaO₂₈[M+Na⁺] 1088.4, found 1088.2.

EXAMPLE 38 Synthesis of Compound 41

The compound 18 (1.3 mg, 1.2 μmol) was subjected to the reaction for 14hours in accordance with the above procedures, to give a desiredcompound 41 (0.8 mg, yield: 80%). The physical data for the resultingcompound are as follows.

¹H-NMR (30° C.) δ5.07 (d, 1H, J=9.5 Hz, GlcNAc1-H-1), 4.91 (s, 1H,Man4′-H-1), 4.76 (s, 1H, Man3-H-1), 4.62 (d, 1H, J=7.8 Hz, GlcNAc2-H-1),4.08 (d, 1H, J=2.9 Hz, Man3-H-2), 2.92 (dd, 1H, J=3.9 Hz, 17.3 Hz,Asn-βCH), 2.83 (dd, 1H, J=7.0 Hz, 17.3 Hz, Asn-βCH), 2.07, 2.01 (each s,each 3H, Ac); MS(Fab) Calcd for C₃₂H₅₅N₄O₂₇[M+H⁺] 863.3, found 863.2.

EXAMPLE 39 Synthesis of Compound 44

The compound 21 (2.3 mg, 1.6 μmol) was subjected to the reaction for 7hours in accordance with the above procedures, to give a desiredcompound 44 (1.6 mg, yield: 84%). The physical data for the resultingcompound are as follows.

¹H-NMR (30° C.) δ5.11 (s, 1H, Man4-H-1), 5.06 (d, 1H,J=9.8 Hz,GlcNAc1-H-1), 4.77 (s, 1H, Man3-H-1), 4.61, 4.57 (each d, each 1H,GlcNAc2,5-H-1), 4.46 (d, 1H, J=7.8 Hz, Gal-H-1), 4.22, 4.18 (each bs,each 1H, Man3,4-H-2), 2.91 (dd, 1H,J=4.1 Hz, 17.3 Hz, Asn-βCH), 2.82(dd, 1H, J=7.0 Hz, 17.3 Hz, Asn-βCH), 2.05, 2.04, 2.01 (each s, each 3H,Ac); MS(Fab) Calcd for C₄₆H₇₈N₅O₃₃[M+H⁺] 1228.5, found 1228.3.

EXAMPLE 40 Synthesis of Compound 45

The compound 22 (1.6 mg, 1.3 μmol) was subjected to the reaction for 14hours in accordance with the above procedures, to give a desiredcompound 45 (1.1 mg, yield: 85%). The physical data for the resultingcompound are as follows.

¹H-NMR (30° C.) δ5.12 (s, 1H, Man4-H-1), 5.07 (d, 1H, J=9.7 Hz,GlcNAc1-H-1), 4.77 (s, 1H, Man3-H-1), 4.61, 4.54 (each d, each 1H,GlcNAc2,5-H-1), 4.22 (d, 1H,J=2.5 Hz, Man3-H-2), 4.18 (dd, 1H,J=1.4 Hz,3.0 Hz, Man4′-H-2), 2.89 (dd, 1H, J=4.3 Hz, 16.9 Hz, Asn-βCH), 2.78 (dd,1H, J=7.5 Hz, 16.9 Hz, Asn-βCH), 2.06, 2.05, 2.01 (each s, each 3H, Ac);MS(Fab) Calcd for C₄₀H₆₇N₅NaO₂₈[M+Na⁺] 1088.4, found 1088.3.

EXAMPLE 41 Synthesis of Compound 46

The compound 23 (1.6 mg, 1.5 μmol) was subjected to the reaction for 14hours in accordance with the above procedures, to give a desiredcompound 46 (1.1 mg, 6.4 μmol, yield: 85%). The physical data for theresulting compound are as follows.

¹H-NMR (30° C.) δ5.10 (s, 1H, Man4-H-1), 5.06 (d, 1H, J=9.5 Hz,GlcNAc1-H-1), 4.77 (s, 1H, Man3-H-1), 4.61 (d, 1H, J=7.3 Hz,GlcNAc2-H-1), 4.22 (d, 1H, J=2.4 Hz, Man3-H-2), 4.07 (dd, 1H, J=1.6 Hz,3.0 Hz, Man4′-H-2), 2.90 (dd, 1H, J=4.3 Hz, 17.0 Hz, Asn-βCH), 2.80 (dd,1H, J=7.0 Hz, 17.2 Hz, Asn-βCH), 2.05, 2.01 (each s, each 3H, Ac);MS(Fab) Calcd for C₃₂H₅₅N₄O₂₃[M+H⁺] 863.3, found 863.3.

EXAMPLE 42 Synthesis of Compound 34

The compound 11 (12.4 mg, 7.5 μmol) was subjected to the reaction for 11hours in accordance with the above procedures, to give a desiredcompound 34 (9.2 mg, yield: 86%). The physical data for the resultingcompound are as follows.

¹H-NMR (30° C.) δ5.11 (s, 1H, Man4-H-1), 5.07 (d, 1H, J=10.0 Hz,GlcNAc1-H-1), 4.91 (s, 1H, Man4′-H-1), 4.77 (s, 1H, Man3-H-1), 4.61 (d,1H, J=6.8 Hz, GlcNAc2-H-1), 4.55 (d, 2H, GlcNAc5,5′-H-1), 4.24 (bs, 1H,Man3-H-2), 4.18 (bs, 1H, Man4′-H-2), 4.10 (bs, 1H, Man4-H-2), 2.80 (dd,1H, J=3.8 Hz, 15.6 Hz, Asn-βCH), 2.63 (dd, 1H, J=8.2 Hz, 15.6 Hz,Asn-βCH), 2.07 (s, 3H, Ac), 2.05 (s, 6H, Ac×2), 2.01 (s, 3H, Ac);MS(Fab) Calcd for C₅₄H₉₀N₆NaO₃₈[M+Na⁺] 1453.5, found 1453.2.

EXAMPLE 43 Synthesis of Compound 35

The compound 12 (12.0 mg, 8.4 μmol) was subjected to the reaction for 11hours in accordance with the above procedures, to give a desiredcompound 35 (7.0 mg, yield: 81%). The physical data for the resultingcompound are as follows.

¹H-NMR (30° C.) δ5.10 (s, 1H, Man4-H-1), 5.07 (d, 1H, J=9.7 Hz,GlcNAc1-H-1), 4.91 (s, 1H, Man4′-H-1), 4.78 (s, 1H, Man3-H-1), 4.61 (d,1H, J=8.0 Hz, GlcNAc2-H-1), 4.25 (bs, 1H, Man3-H-2), 4.06 (bs, 1H,Man4′-H-2), 3.97 (bs, 1H, Man4-H-2), 2.79 (dd, 1H, J=5.0 Hz, 17.0 Hz,Asn-βCH), 2.61 (dd, 1H, J=7.3 Hz, 17.0 Hz, Asn-βCH), 2.07, 2.01 (each s,each 3H, Ac); MS(Fab) Calcd for C₃₈H₆₅N₄O₂₈[M+H⁺] 1025.4, found 1025.2.

EXAMPLE 44 Synthesis and Isolation of Compounds 76 and 77

The compounds 2 and 6 (5.0 mg, 2.2 μmol) were dissolved in 220 μL ofwater, and 100 μL of a 22 mM aqueous cesium carbonate was added theretoto adjust its pH to 7.0. This solution was lyophilized. Four-hundred andthirty microliters of N,N-dimethylformamide was added to the solidobtained after drying, and further 20 μL of a 6.6 μmol benzylbromide/N,N-dimethylformamide solution was added thereto. This solutionwas stirred under argon atmosphere. After 48 hours, the disappearance ofthe starting material was confirmed by TLC (eluent: 1MNH₄OAc:isopropanol=2:1), and thereafter 4.4 mL of diethyl ether wasadded to the solution to allow the compound to precipitate therefrom.The precipitated sugar chains were filtered, and the residual sugarchain was dissolved in water and lyophilized. The residue after thelyophilization was purified by fractional HPLC (YMC Packed ColumnD-ODS-5 S-5 120A ODS No. 2020178, 20×250 mm, eluent: 50 mM aqueousammonium acetate:acetonitrile=78:22, flow rate: 4 mL/min), and acompound 77 was eluted after 88 minutes and a compound 76 was elutedafter 91 minutes. The fractions were collected, and further desalted onan ODS column (Cosmosil 75C₁₈-OPN, 15×100 mm, eluted first with 50 mL ofH₂O and then with 25% acetonitrile), to give a desired compound 76 in anamount of 1.6 mg and a desired compound 77 in an amount of 1.8 mg. Thephysical data for the resulting compound are as follows.

Data for Compound 76

¹H-NMR (30° C.) δ7.92 (d, 2H, J=7.5 Hz, Fmoc), 7.71 (d, 2H, J=7.5 Hz,Fmoc), 7.53–7.40 (m, 9H, Fmoc, —CH₂-Ph), 5.38 (d, 1H, J=12.1 Hz, —CH ²-Ph), 5.31 (d, 1H, J=12.1 Hz, —CH ² -Ph), 5.12 (s, 1H, Man4-H-1), 4.99(d, 1H, J=9.5 Hz, GlcNAc1-H-1), 4.92 (s, 1H, Man4′-H-1), 4.76 (s, 1H,Man3-H-1), 4.58 (m, 3H, GlcNAc2,5,5′-H-1), 4.47 (d, 1H, J=7.9 Hz,Gal6′-H-1), 4.33 (d, 1H, J=7.9 Hz, Gal6-H-1), 4.24 (bs, 1H, Man3-H-2),4.19 (bs, 1H, Man4′-H-2),4.11 (bs, 1H, Man4-H-2), 2.72 (bd, 1H,Asn-βCH), 2.68 (dd, 1H, J=4.6 Hz, 12.7 Hz, NeuAc7-H-3_(eq)), 2.52 (dd,1H, J=8.7 Hz, 15.0 Hz, Asn-βCH), 2.07, 2.04, 2.03, 2.02, 1.89 (each s,each 3H, Ac), 1.84 (dd, 1H, J=12.7 Hz, 12.7 Hz, NeuAc7-H3_(ax)); MS(Fab)Calcd for C₉₉H₁₄₃N₁₇NaO₅₈[M+H⁺] 2380.8, found 2380.0.

Data for Compound 77

¹H-NMR (30° C.) δ7.91 (d, 2H, J=7.5 Hz, Fmoc), 7.71 (d, 2H, J=7.5 Hz,Fmoc), 7.53–7.41 (m, 9H, Fmoc, —CH₂-Ph), 5.37 (d, 1H, J=12.1 Hz, —CH ²-Ph), 5.31 (d, 1H, J=12.1 Hz, —CH ² -Ph), 5.12 (s, 1H, Man4-H-1), 4.99(d, 1H, J=9.8 Hz, GlcNAc1-H-1), 4.93 (s, 1H, Man4′-H-1), 4.76 (s, 1H,Man3-H-1), 4.58 (m, 3H, GlcNAc2,5,5′-H-1), 4.46 (1H, d, J=7.8 Hz,Gal6-H-1), 4.33 (d, 1H, J=7.8 Hz, Gal6′-H-1), 4.24 (bs, 1H, Man3-H-2),4.19 (bs, 1H, Man4′-H-2), 4.11 (bs, 1H, Man4-H-2), 2.72 (bd, 1H,Asn-βCH), 2.68 (dd, 1H, J=4.8 Hz, 13.0 Hz, NeuAc7-H-3_(eq)), 2.52 (bdd,1H, J=9.7 Hz, 14.1 Hz, Asn-βCH), 2.06, 2.05, 2.04, 2.02, 1.89 (each s,each 3H, Ac), 1.84 (dd, 1H, J=13.0 Hz, 13.0 Hz, NeuAc7-H-3_(ax));MS(Fab) Calcd for C₉₉H₁₄₃N₇NaO₅₈[M+H⁺] 2380.8, found 2380.5.

EXAMPLE 45 Synthesis of Compound 78

A cooled aqueous solution of the compound 1 (20 mg) was applied onto aDowex-50 W×8 (H⁺) column (φ 0.5 cm×5 cm) chilled at 4° C., and theeluted aqueous solution was lyophilized. The resulting lyophilizedproduct was dissolved in a chilled water at 4° C., an aqueous Cs₂CO₃(2.5 mg/1 ml) was added thereto to adjust a pH of the aqueous solutionto 5 to 6, and the aqueous solution was then lyophilized. TheFmoc-disialo-sugar chain sample after the lyophilization was dissolvedin a dry DMF (1.3 ml), and benzyl bromide (5.1 μl) was added theretowith stirring the mixture at room temperature for 45 minutes under argongas stream. The termination of the reaction was confirmed by TLC,thereafter the reaction solution was cooled to 0° C., and 10 ml ofdiethyl ether was added to the reaction solution to precipitate thedesired product. This substance was filtered with a filter paper.Distilled water was added to the residual desired product, and eluted asa filtrate, and subsequently concentrated under a reduced pressure. Theresulting residue was purified by applying to an ODS column (φ 1.6 cm×14cm, eluent: H₂O→40% aqueous MeOH), to give a compound 78 (18.2 mg,yield: 85%). The physical data for the resulting compound 78 are asfollows.

¹H-NMR (30° C.) 7.90 (d, 2H, Fmoc), 7.70 (d, 2H, Fmoc), 7.53–7.40 (m,9H, Bn, Fmoc), 5.36 (d, 2H, J=11.6 Hz, CH₂), 5.30 (d, 2H, J=11.6 Hz,CH₂), 5.12 (s, 1H, Man4-H,), 4.99 (d, 1H, J=9.7 Hz, GlcNAc1-H₁), 4.93(s, 1H, Man4′-H₁), 4.75 (s, 1H, Man3-H₁), 4.57 (m, 3H, GlcNAc2-H₁,GlcNAc5,5′-H,), 4.32 (d, 2H, Gal6,6′-H₁), 4.24 (d, 1H, Man3-H₂), 4.18(d, 1H, Man4′-H₂), 4.10 (1H, d, Man4-H₂) 2.72 (bd, 1H, Asn-βCH), 2.67(dd, 2H, NeuAc7,7′-H_(3eq)), 2.51 (bdd, 1H, Asn-βCH), 2.06 (s, 3H, Ac),2.03, 2.01 (each s, each 6H, Ac×2), 1.89 (s, 3H, Ac), 1.83 (2H, dd,J=12.2, 12.2 Hz, NeuAc7,7′-H_(3ax)); HRMS Calcd forC₁₁₇H₁₆₅N₈Na₂O₆₆[M+Na⁺] 2783.9597, found 2783.9501.

INDUSTRIAL APPLICABILITY

According to the present invention, various sugar chains having desiredsugar chain structures can be obtained in large amounts veryconveniently. Therefore, there is expected to be utilized fortherapeutic agents against cancer, inflammation, influenza and the like.Especially, the sugar chain asparagine derivative and sugar chainasparagine which can be obtained in the present invention are excellentin safety without any risks such as admixing of toxic substances duringthe preparation processes.

The invention claimed is:
 1. A process for preparing a sugar chainasparagine derivative derived from a sugar chain asparagine, comprisingthe steps of: (a) introducing a fat-soluble protecting group selectedfrom the group consisting of Fmoc and Boc groups into the sugar chainaspargine contained in a mixture of one or more sugar chain asparagines,to give a mixture of sugar chain asparagine derivatives; and (b)subjecting the mixture of sugar chain asparagine derivatives or amixture obtainable by hydrolyzing a sugar chain asparagine derivativecontained in the mixture of sugar chain asparagine derivatives tochromatography, to separate each of the sugar chain asparaginederivatives therefrom.
 2. The process according to claim 1, furthercomprising the step of (b′): hydrolyzing the sugar chain asparaginederivative separated in step (b) with a glycosidase.
 3. The processaccording to claim 1 or 2, wherein the mixture of one or more sugarchain asparagines comprises a compound of the following formula, inwhich Asn denotes asparagine:

and/or a compound having one or more deletions of sugar residues in theabove compound.
 4. The process according to any one of claims 1 to 3,wherein the fat-soluble protecting group is fluorenylmethoxycarbonyl(Fmoc) group.
 5. The process according to any one of claims 1 to 3,wherein the step (a) is a step of introducing Fmoc group into the sugarchain aspargine contained in a mixture of one or more sugar chainasparagines having a sialic acid residue at a non-reducing terminal, andintroducing benzyl group into the sialic acid residue, to give a mixtureof sugar chain asparagine derivatives.
 6. A process for isolating asugar chain aspargine from a mixture, comprising the steps of: (a)introducing a fat-soluble protecting group selected from the groupconsisting of Fmoc and Boc groups into a sugar chain aspargine containedin a mixture of one or more sugar chain asparagines, to give a mixtureof sugar chain asparagine derivatives; (b) subjecting the mixture ofsugar chain asparagine derivatives or a mixture obtainable byhydrolyzing a sugar chain asparagine derivative contained in the mixtureof sugar chain asparagine derivatives to chromatography, to separateeach of the sugar chain asparagine derivatives therefrom; and (c)removing the protecting group from the sugar chain asparagine derivativeseparated in the step (b), to give the sugar chain asparagine.
 7. Theprocess according to claim 6, further comprising the step of: (b′)hydrolyzing the sugar chain asparagine derivative separated in step (b)with a glycosidase; and/or (c′) hydrolyzing the sugar chain asparagineobtained in step (c) with a glycosidase.
 8. The process according toclaim 6 or 7, wherein the mixture of one or more sugar chain asparaginescomprises a compound of the following formula, in which Asn denotesasparagine:

and/or a compound having one or more deletions of sugar residues in theabove compound.
 9. The process according to any one of claims 6 to 8,wherein the fat-soluble protecting group is Fmoc group.
 10. The processaccording to any one of claims 6 to 8, wherein the step (a) is a step ofintroducing Fmoc group into the sugar chain aspargine contained in amixture of one or more sugar chain asparagines having a sialic acidresidue at a non-reducing terminal, and introducing benzyl group intothe sialic acid residue, to give a mixture of sugar chain asparaginederivatives.
 11. A process for preparing a sugar chain from a mixture ofsugar chain asparagines, comprising the steps of: (a) introducing afat-soluble protecting group selected from the group consisting of Fmocand Boc groups into a sugar chain aspargine contained in a mixture ofone or more sugar chain asparagines, to give a mixture of sugar chainasparagine derivatives; (b) subjecting the mixture of sugar chainasparagine derivatives or a mixture obtainable by hydrolyzing a sugarchain asparagine derivative contained in the mixture of sugar chainasparagine derivatives to chromatography, to separate each of the sugarchain asparagine derivatives therefrom; (c) removing the protectinggroup from the sugar chain asparagine derivative separated in the step(b), to give a sugar chain asparagine; and (d) removing an asparagineresidue from the sugar chain asparagine obtained in the step (c), togive the sugar chain.
 12. The process claim 11, further comprising thestep of: (b′) hydrolyzing the sugar chain asparagine derivativeseparated in step (b) with a glycosidase; and/or (c′) hydrolyzing thesugar chain asparagine obtained in step (c) with a glycosidase; and/or(d′) hydrolyzing the sugar chain obtained in step (d) with aglycosidase.
 13. The process according to claim 11 or 12, wherein themixture of one or more sugar chain asparagines comprises a compound ofthe following formula, in which Asn denotes asparagine:

and/or a compound having one or more deletions of sugar residues in theabove compound.
 14. The process according to any one of claims 11 to 13,wherein the fat-soluble protecting group is Fmoc group.
 15. The processaccording to any one of claims 11 to 13, wherein the step (a) is a stepof introducing Fmoc group into the sugar chain aspargine contained in amixture of one or more sugar chain asparagines having a sialic acidresidue at a non-reducing terminal, and introducing benzyl group intothe sialic acid residue, to give a mixture of sugar chain asparaginederivatives.
 16. A sugar chain asparagine derivative having the generalformula:

wherein Asn is asparagine, R¹ and R², which may be identical ordifferent, are H,

with proviso that a case where R¹ and R² are both H,

is excluded.
 17. A sugar chain asparagine derivative having the generalformula:

wherein one of R^(x) and R^(y) is

and the other is H,


18. A sugar chain asparagine having the general formula:

wherein Asn is asparagine, R³ and R⁴, which may be identical ordifferent, are H,

with proviso that (i) a case where R³ and R⁴ are the same, (ii) a casewhere R3 is

and R⁴ is H or

and (iii) a case where R³ is H and R⁴ is

are excluded.
 19. A sugar chain having the general formula:

wherein R⁵ and R⁶, which may be identical or different, are H,

with proviso that (i) a case where R⁵ and R⁶ are the same, (ii) a casewhere one of R⁵ and R⁶ is

and the other is H or

and (iii) a case where R⁵ is

and R⁶ is

are excluded.